The zebrafish (Danio rerio) is an important model organism for studying development and human disease. The zebrafish has an excellent reference genome and the functions of hundreds of genes have been tested using both forward and reverse genetic approaches. Recent years have seen an increasing number of large-scale mutagenesis projects and the number of mutants or gene knockouts in zebrafish has increased rapidly, including for the first time conditional knockout technologies. In addition, targeted mutagenesis techniques such as zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short sequences (CRISPR) or CRISPR-associated (Cas), have all been shown to effectively target zebrafish genes as well as the first reported germline homologous recombination, further expanding the utility and power of zebrafish genetics. Given this explosion of mutagenesis resources, it is now possible to perform systematic, high-throughput phenotype analysis of all zebrafish gene knockouts.
zebrafish; mutagenesis; phenotyping; resources; knockouts
Defects in the initial establishment of cardiogenic cell fate are likely to contribute to pervasive human congenital cardiac abnormalities. However, the molecular underpinnings of nascent cardiac fate induction have proven difficult to decipher. In this review we explore the participation of extracellular, cellular and nuclear factors in comprehensive specification networks. At each level, we elaborate on insights gained through the study of cardiogenesis in the invertebrate chordate Ciona intestinalis and propose productive lines of future research. In-depth discussion of pre-cardiac induction is intended to serve as a paradigm, illustrating the potential use of Ciona to elucidate comprehensive networks underlying additional aspects of chordate cardiogenesis, including directed migration and subspecification of cardiac and pharyngeal lineages.
Mesp; FGF; gene regulatory networks; extracellular matrix; cardiac induction; chordate evolution
Three interlocking problems in gene regulation are: how to explain genome-wide targeting of transcription factors in different cell types, how prior transcription factor action can establish an ‘epigenetic state’ that changes the options for future transcription factor action, and how directly a sequence of developmental decisions can be memorialized in a hierarchy of repression structures applied to key genes of the ‘paths not taken’. This review uses the finely staged process of T-cell lineage commitment as a test case in which to examine how changes in developmental status are reflected in changes in transcription factor expression, transcription factor binding distribution across genomic sites, and chromatin modification. These are evaluated in a framework of reciprocal effects of previous chromatin structure features on transcription factor access and of transcription factor binding on other factors and on future chromatin structure.
DNA binding; histone methylation; DNA methylation; lineage commitment; repression; hematopoiesis
Our understanding of fungal cellulose degradation has shifted dramatically in the past few years with the characterization of a new class of secreted enzymes, the lytic polysaccharide monooxygenases (LPMO). After a period of intense research covering structural, biochemical, theoretical and evolutionary aspects, we have a picture of them as wedge-like copper-dependent metalloenzymes that on reduction generate a radical copper-oxyl species, which cleaves mainly crystalline cellulose. The main biological function lies in the synergism of fungal LPMOs with canonical hydrolytic cellulases in achieving efficient cellulose degradation. Their important role in cellulose degradation is highlighted by the wide distribution and often numerous occurrences in the genomes of almost all plant cell-wall degrading fungi. In this review, we provide an overview of the latest achievements in LPMO research and consider the open questions and challenges that undoubtedly will continue to stimulate interest in this new and exciting group of enzymes.
AA9; GH61; cellobiose dehydrogenase; oxidative cellulose degradation
Microarray technologies provide high-throughput analysis of genes that are differentially expressed in humans and other species, and thereby provide a means to measure how biological systems are altered during development or disease states. Within, we review how high-throughput genomic technologies have increased our understanding about the molecular complexity of breast cancer, identified distinct molecular phenotypes and how they can be used to increase the accuracy of predicted clinical outcome.
breast cancer; microarray; genomics; tumor; histology
Primary metabolism affects all phenotypical traits of filamentous fungi. Particular examples include reacting to extracellular stimuli, producing precursor molecules required for cell division and morphological changes as well as providing monomer building blocks for production of secondary metabolites and extracellular enzymes. In this review, all annotated genes from four Aspergillus species have been examined. In this process, it becomes evident that 80–96% of the genes (depending on the species) are still without verified function. A significant proportion of the genes with verified metabolic functions are assigned to secondary or extracellular metabolism, leaving only 2–4% of the annotated genes within primary metabolism. It is clear that primary metabolism has not received the same attention in the post-genomic area as many other research areas—despite its role at the very centre of cellular function. However, several methods can be employed to use the metabolic networks in tandem with comparative genomics to accelerate functional assignment of genes in primary metabolism. In particular, gaps in metabolic pathways can be used to assign functions to orphan genes. In this review, applications of this from the Aspergillus genes will be examined, and it is proposed that, where feasible, this should be a standard part of functional annotation of fungal genomes.
Aspergillus; primary metabolism; functional genomics; metabolic networks
The use of genetically isolated populations can empower next-generation association studies. In this review, we discuss the advantages of this approach and review study design and analytical considerations of genetic association studies focusing on isolates. We cite successful examples of using population isolates in association studies and outline potential ways forward.
isolated populations; rare variants; complex disease; genetic association studies
Our understanding of transcriptional gene regulation has dramatically increased over the past decades, and many regulators of gene expression, such as transcription factors, have been analyzed extensively. Additionally, in recent years, deeper insights into the physiological roles of RNA have been obtained. More precisely, splicing, polyadenylation, various modifications, localization and the translation of messenger RNAs (mRNAs) are regulated by their interaction with RNA-binding proteins (RBPs). New technologies now enable the analysis of this regulation at different levels. A technique known as ultraviolet (UV) cross-linking and immunoprecipitation (CLIP) allows us to determine physical protein–RNA interactions on a genome-wide scale. UV cross-linking introduces covalent bonds between interacting RBPs and RNAs. In combination with immunoprecipitation and deep sequencing techniques, tens of millions of short reads (representing bound RNAs by an RBP of interest) are generated and are used to characterize the regulatory network mediated by an RBP. Other methods, such as mass spectrometry, can also be used for characterization of cross-linked RBPs and RNAs instead of CLIP methods. In this review, we discuss experimental and computational methods for the generation and analysis of CLIP data. The computational methods include short-read alignment, annotation and RNA-binding motif discovery. We describe the challenges of analyzing CLIP data and indicate areas where improvements are needed.
Next-generation sequencing; cross-linking and immunoprecipitation; posttranscriptional gene regulation; RNA-binding motif discovery
The assessment of genome function requires a mapping between genome-derived entities and biochemical reactions, and the biomedical literature represents a rich source of information about reactions between biological components. However, the increasingly rapid growth in the volume of literature provides both a challenge and an opportunity for researchers to isolate information about reactions of interest in a timely and efficient manner. In response, recent text mining research in the biology domain has been largely focused on the identification and extraction of ‘events’, i.e. categorised, structured representations of relationships between biochemical entities, from the literature. Functional genomics analyses necessarily encompass events as so defined. Automatic event extraction systems facilitate the development of sophisticated semantic search applications, allowing researchers to formulate structured queries over extracted events, so as to specify the exact types of reactions to be retrieved. This article provides an overview of recent research into event extraction. We cover annotated corpora on which systems are trained, systems that achieve state-of-the-art performance and details of the community shared tasks that have been instrumental in increasing the quality, coverage and scalability of recent systems. Finally, several concrete applications of event extraction are covered, together with emerging directions of research.
text mining; event extraction; semantic annotation; semantic search
Understanding the forces that shape patterns of genetic variation across the genome is a major aim in evolutionary genetics. An emerging insight from analyses of genome-wide polymorphism and divergence data is that selection on linked sites can have an important impact on neutral genetic variation. However, in contrast to Drosophila, which exhibits a signature of recurrent hitchhiking, many plant genomes studied so far seem to mainly be affected by background selection. Moreover, many plants do not exhibit classic signatures of linked selection, such as a correlation between recombination rate and neutral diversity. In this review, I discuss the impact of genome architecture and mating system on the expected signature of linked selection in plants and review empirical evidence for linked selection, with a focus on plant model systems. Finally, I discuss the implications of linked selection for inference of demographic history in plants.
hitchhiking; genetic draft; recombination rate; genome size; FST; demographic inference
In this review, we discuss a strategy to bring genomics and proteomics into single cells by super-resolution microscopy. The basis for this new approach are the following: given the 10 nm resolution of a super-resolution microscope and a typical cell with a size of (10 µm)3, individual cells contain effectively 109 super-resolution pixels or bits of information. Most eukaryotic cells have 104 genes and cellular abundances of 10–100 copies per transcript. Thus, under a super-resolution microscope, an individual cell has 1000 times more pixel volume or information capacities than is needed to encode all transcripts within that cell. Individual species of mRNA can be uniquely identified by labeling them each with a distinct combination of fluorophores by fluorescence in situ hybridization. With at least 15 fluorophores available in super-resolution, hundreds of genes in can be barcoded with a three-color barcode (3C15 = 455). These calculations suggest that by combining super-resolution microscopy and barcode labeling, single cells can be turned into informatics platforms denser than microarrays and that molecular species in individual cells can be profiled in a massively parallel fashion.
super-resolution microscopy; systems biology; single cells; single-molecule FISH
All organisms have to safeguard the integrity of their genome to prevent malfunctioning and oncogenic transformation. Sophisticated DNA damage response mechanisms have evolved to detect and repair genomic lesions. With the emergence of live-cell microscopy of individual cells, we now begin to appreciate the complex spatiotemporal kinetics of the DNA damage response and can address the causes and consequences of the heterogeneity in the responses of genetically identical cells. Here, we highlight key discoveries where live-cell imaging has provided unprecedented insights into how cells respond to DNA double-strand breaks and discuss the main challenges and promises in using this technique.
live-cell imaging; single cell; DNA damage; fluorescence microscopy; dynamics
Epigenetic modifications are implicated in the maintenance and regulation of transcriptional memory by marking genes that were previously transcribed to facilitate transmission of these expression patterns through cell division. During germline specification and maintenance, extensive epigenetic modifications are acquired. Yet somehow at fertilization, the fusion of the highly differentiated sperm and egg results in formation of the totipotent zygote. This massive change in cell fate implies that the selective erasure and maintenance of epigenetic modifications at fertilization may be critical for the re-establishment of totipotency. In this review, we discuss recent studies that provide insight into the extensive epigenetic reprogramming that occurs around fertilization and the mechanisms that may be involved in the re-establishment of totipotency in the embryo.
Researchers in the field of epigenomics are developing more nuanced understandings of biological complexity, and exploring the multiple pathways that lead to phenotypic expression. The concept of degeneracy—referring to the multiple pathways that a system recruits to achieve functional plasticity—is an important conceptual accompaniment to the growing body of knowledge in epigenomics. Distinct from degradation, redundancy and dilapidation; degeneracy refers to the plasticity of traits whose function overlaps in some environments, but diverges in others. While a redundant system is composed of repeated identical elements performing the same function, a degenerate system is composed of different elements performing similar or overlapping functions. Here, we describe the degenerate structure of gene regulatory systems from the basic genetic code to flexible epigenomic modifications, and discuss how these structural features have contributed to organism complexity, robustness, plasticity and evolvability.
epigenetic code; pluripotentiality; robustness; redundancy; DNA methylation; histone modifications; social insect; honey bee
The precise developmental map of the Caenorhabditis elegans cell lineage, as well as a complete genome sequence and feasibility of genetic manipulation make this nematode species highly attractive to study the role of epigenetics during development. Genetic dissection of phenotypical traits, such as formation of egg-laying organs or starvation-resistant dauer larvae, has illustrated how chromatin modifiers may regulate specific cell-fate decisions and behavioral programs. Moreover, the transparent body of C. elegans facilitates non-invasive microscopy to study tissue-specific accumulation of heterochromatin at the nuclear periphery. We also review here recent findings on how small RNA molecules contribute to epigenetic control of gene expression that can be propagated for several generations and eventually determine longevity.
Caenorhabditis elegans; chromatin organization; longevity; organogenesis; small RNA; transcriptional silencing
In this review, we present an overview of the recent advances of genomic technologies applied to studies of fish species belonging to the superclass of Osteichthyes (bony fish) with a major emphasis on the infraclass of Teleostei, also called teleosts. This superclass that represents more than 50% of all known vertebrate species has gained considerable attention from genome researchers in the last decade. We discuss many examples that demonstrate that this highly deserved attention is currently leading to new opportunities for answering important biological questions on gene function and evolutionary processes. In addition to giving an overview of the technologies that have been applied for studying various fish species we put the recent advances in genome research on the model species zebrafish and medaka in the context of its impact for studies of all fish of the superclass of Osteichthyes. We thereby want to illustrate how the combined value of research on model species together with a broad angle perspective on all bony fish species will have a huge impact on research in all fields of fundamental science and will speed up applications in many societally important areas such as the development of new medicines, toxicology test systems, environmental sensing systems and sustainable aquaculture strategies.
fish models; teleosts; genomics; aquaculture; next-generation sequencing; zebrafish; medaka
The development of gene targeting approaches has had a tremendous impact on the functional analysis of the mouse genome. A specific application of this technique has been the adaptation of the bacteriophage P1 Cre/loxP site-specific recombinase system which allows for the precise recombination between two loxP sites, resulting in deletion or inversion of the intervening sequences. Because of the efficiency of this system, it can be applied to conditional deletions of relatively short coding sequences or regulatory elements but also to more extensive chromosomal rearrangement strategies. Both mechanistic and functional studies of genomic imprinting have benefited from the development of the Cre/loxP technology. Since imprinted genes within large chromosomal regions are regulated by the action of cis-acting sequences known as imprinting centres, chromosomal engineering approaches are particularly well suited to the elucidation of long-range mechanisms controlling the imprinting of autosomal genes. Here we review the applications of the Cre/loxP technology to the study of genomic imprinting, highlight important insights gained from these studies and discuss future directions in the field.
PMID: 20601421 CAMSID: cams3612
genomic imprinting; Cre-loxP system; chromosome engineering
Studies of large imprinted clusters, such as the Gnas locus, have revealed much about the significance of DNA methylation, transcription and other factors in the establishment and maintenance of imprinted gene expression. However, the complexity of such loci can make manipulating them and interpreting the results challenging. We review here a distinct class of imprinted genes, which have arisen by retrotransposition, and which have the potential to be used as models for the dissection of the fundamental features and mechanisms required for imprinting. They are also of interest in their own right, generating diversity in the transcriptome and providing raw material upon which selection can act.
retrotransposition; retrogenes; genomic imprinting; epigenetics
In this review, we provide a detailed overview of studies on the elusive sex determination (SD) and gonad differentiation mechanisms of zebrafish (Danio rerio). We show that the data obtained from most studies are compatible with polygenic sex determination (PSD), where the decision is made by the allelic combinations of several loci. These loci are typically dispersed throughout the genome, but in some teleost species a few of them might be located on a preferential pair of (sex) chromosomes. The PSD system has a much higher level of variation of SD genotypes both at the level of gametes and the sexual genotype of individuals, than that of the chromosomal sex determination systems. The early sexual development of zebrafish males is a complicated process, as they first develop a ‘juvenile ovary’, that later undergoes a transformation to give way to a testis. To date, three major developmental pathways were shown to be involved with gonad differentiation through the modulation of programmed cell death. In our opinion, there are more pathways participating in the regulation of zebrafish gonad differentiation/transformation. Introduction of additional powerful large-scale genomic approaches into the analysis of zebrafish reproduction will result in further deepening of our knowledge as well as identification of additional pathways and genes associated with these processes in the near future.
polygenic sex determination; sex chromosome; gonad differentiation; teleost; fish; Danio rerio
Proper differentiation of naïve T helper cells into functionally distinct subsets is of critical importance to human health. Consequently, the process is tightly controlled by a complex intracellular signalling network. To dissect the regulatory principles of this network, immunologists have early on embraced system-wide transcriptomics tools, leading to identification of large panels of potential regulatory factors. In contrast, the use of proteomics approaches in T helper cell research has been notably rare, and to this date relatively few high-throughput datasets have been reported. Here, we discuss the importance of such research and envision the possibilities afforded by mass spectrometry-based proteomics in the near future.
T helper cell; systems biology; proteomics; mass spectrometry; transcriptomics; T cell activation
Drosophilists have identified many, or perhaps most, of the key regulatory genes determining sex using classical genetics, however, regulatory genes must ultimately result in the deployment of the genome in a quantitative manner, replete with complex interactions with other regulatory pathways. In the last decade, genomics has provided a rich picture of the transcriptional profile of the sexes that underlies sexual dimorphism. The current challenge is linking transcriptional profiles with the regulatory genes. This will be a complex synthesis, but the prospects for progress are outstanding.
drosophila; transcriptome; sex determination; effector; selector
Researchers have now had access to the fully sequenced Drosophila melanogaster genome for over a decade, and the sequenced genomes of 11 additional Drosophila species have been available for almost 5 years, with more species’ genomes becoming available every year [Adams MD, Celniker SE, Holt RA, et al. The genome sequence of Drosophila melanogaster. Science 2000;287:2185–95; Clark AG, Eisen MB, Smith DR, et al. Evolution of genes and genomes on the Drosophila phylogeny. Nature 2007;450:203–18]. Although the best studied of the D. melanogaster transcription factors (TFs) were cloned before sequencing of the genome, the availability of sequence data promised to transform our understanding of TFs and gene regulatory networks. Sequenced genomes have allowed researchers to generate tools for high-throughput characterization of gene expression levels, genome-wide TF localization and analyses of evolutionary constraints on DNA elements across multiple species. With an estimated 700 DNA-binding proteins in the Drosophila genome, it will be many years before each potential sequence-specific TF is studied in detail, yet the last decade of functional genomics research has already impacted our view of gene regulatory networks and TF DNA recognition.
Drosophila; transcription factor; genomics; enhancer; Zelda
Many aspects of gene regulation are mediated by RNA molecules. However, regulatory RNAs have remained elusive until very recently. At least three types of small regulatory RNAs have been characterized in Drosophila: microRNAs (miRNAs), piwi-interacting RNAs and endogenous siRNAs. A fourth class of regulatory RNAs includes known long non-coding RNAs such as roX1 or bxd. The initial sequencing of the Drosophila melanogaster genome has served as a scaffold to study the transcriptional profile of an animal, revealing the complexities of the function and biogenesis of regulatory RNAs. The comparative analysis of 12 Drosophila genomes has been crucial for the study of microRNA evolution. However, comparative genomics of other RNA regulators is confounded by technical problems: genomic loci are poorly conserved and frequently encoded in the heterochromatin. Future developments in genome sequencing and population genomics in Drosophila will continue to shed light on the conservation, evolution and function of regulatory RNAs.
Non-coding RNA; miRNA; piRNA; siRNA; transposable elements; gene regulation
Inflammation is a fundamental response of the immune system whose successful termination involves the elimination of the invading pathogens, the resolution of inflammation and the repair of the local damaged tissue. In this context, the interleukin 10 (IL-10)-mediated anti-inflammatory response (AIR) represents an essential homeostatic mechanism that controls the degree and duration of inflammation. Here, we review recent work on the mechanistic characterization of the IL-10-mediated AIR on multiple levels: from the cataloguing of the in vivo genomic targets of STAT3 (the transcription factor downstream of IL-10) to the identification of specific co-factors that endow STAT3 with genomic-binding specificity, and how genomic and computational methods are being used to elucidate the regulatory mechanisms of this essential physiological response in macrophages.
IL-10; JAK1; STAT3; anti-inflammatory response; macrophages; transcriptional regulatory modules; bioinformatics
The differentiation of CD4 helper T cells into specialized effector lineages has provided a powerful model for understanding immune cell differentiation. Distinct lineages have been defined by differential expression of signature cytokines and the lineage-specifying transcription factors necessary and sufficient for their production. The traditional paradigm of differentiation towards Th1 and Th2 subtypes driven by T-bet and GATA3, respectively, has been extended to incorporate additional T cell lineages and transcriptional regulators. Technological advances have expanded our view of these lineage-specifying transcription factors to the whole genome and revealed unexpected interplay between them. From these data, it is becoming clear that lineage specification is more complex and plastic than previous models might have suggested. Here, we present an overview of the different forms of transcription factor interplay that have been identified and how T cell phenotypes arise as a product of this interplay within complex regulatory networks. We also suggest experimental strategies that will provide further insight into the mechanisms that underlie T cell lineage specification and plasticity.
T cell; transcription factor; lineage-specification; cell differentiation; plasticity; enhancer