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
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
Plasmodium falciparum is an obligate intracellular parasite and the leading cause of severe malaria responsible for tremendous morbidity and mortality particularly in sub-Saharan Africa. Successful completion of the P. falciparum genome sequencing project in 2002 provided a comprehensive foundation for functional genomic studies on this pathogen in the following decade. Over this period, a large spectrum of experimental approaches has been deployed to improve and expand the scope of functionally annotated genes. Meanwhile, rapidly evolving methods of systems biology have also begun to contribute to a more global understanding of various aspects of the biology and pathogenesis of malaria. Herein we provide an overview on metabolic modelling, which has the capability to integrate information from functional genomics studies in P. falciparum and guide future malaria research efforts towards the identification of novel candidate drug targets.
Plasmodium falciparum; central carbon metabolism; systems biology; flux-balance analysis; constraint-based modelling; in silico gene essentiality
Methylation of histone H3 at lysine 4 (H3K4) is a conserved feature of active chromatin catalyzed by methyltransferases of the SET1-family (SET1A, SET1B, MLL1, MLL2, MLL3 and MLL4 in humans). These enzymes participate in diverse gene regulatory networks with a multitude of known biological functions, including direct involvement in several human disease states. Unlike most lysine methyltransferases, SET1-family enzymes are only fully active in the context of a multi-subunit complex, which includes a protein module comprised of WDR5, RbBP5, ASH2L and DPY-30 (WRAD). These proteins bind in close proximity to the catalytic SET domain of SET1-family enzymes and stimulate H3K4 methyltransferase activity. The mechanism by which WRAD promotes catalysis involves elements of allosteric control and possibly the utilization of a second H3K4 methyltransferase active site present within WRAD itself. WRAD components also engage in physical interactions that recruit SET1-family proteins to target sites on chromatin. Here, the known molecular mechanisms through which WRAD enables the function of SET1-related enzymes will be reviewed.
SET1; MLL; WDR5; RbBP5; ASH2L; DPY-30
Epigenetic genome marking and chromatin regulation are central to establishing tissue-specific gene expression programs, and hence to several biological processes. Until recently, the only known epigenetic mark on DNA in mammals was 5-methylcytosine, established and propagated by DNA methyltransferases and generally associated with gene repression. All of a sudden, a host of new actors—novel cytosine modifications and the ten eleven translocation (TET) enzymes—has appeared on the scene, sparking great interest. The challenge is now to uncover the roles they play and how they relate to DNA demethylation. Knowledge is accumulating at a frantic pace, linking these new players to essential biological processes (e.g. cell pluripotency and development) and also to cancerogenesis. Here, we review the recent progress in this exciting field, highlighting the TET enzymes as epigenetic DNA modifiers, their physiological roles, and their functions in health and disease. We also discuss the need to find relevant TET interactants and the newly discovered TET–O-linked N-acetylglucosamine transferase (OGT) pathway.
epigenetics; DNA methylation; hydroxymethylation; TET proteins; OGT
There is an increasing availability of complete or draft genome sequences for microbial organisms. These data form a potentially valuable resource for genotype–phenotype association and gene function prediction, provided that phenotypes are consistently annotated for all the sequenced strains. In this review, we address the requirements for successful gene-trait matching. We outline a basic protocol for microbial functional genomics, including genome assembly, annotation of genotypes (including single nucleotide polymorphisms, orthologous groups and prophages), data pre-processing, genotype–phenotype association, visualization and interpretation of results. The methodologies for association described herein can be applied to other data types, opening up possibilities to analyze transcriptome–phenotype associations, and correlate microbial population structure or activity, as measured by metagenomics, to environmental parameters.
genotype–phenotype association; genome-wide association studies; functional genomics; microbial genomics; random forest
Epigenetic memory represents a natural mechanism whereby the identity of a cell is maintained through successive cell cycles, allowing the specification and maintenance of differentiation during development and in adult cells. Cancer is a loss or reversal of the stable differentiated state of adult cells and may be mediated in part by epigenetic changes. The identity of somatic cells can also be reversed experimentally by nuclear reprogramming. Nuclear reprogramming experiments reveal the mechanisms required to activate embryonic gene expression in adult cells and thus provide insight into the reversal of epigenetic memory. In this article, we will introduce epigenetic memory and the mechanisms by which it may operate. We limit our discussion primarily to the context of nuclear reprogramming and briefly discuss the relevance of memory and reprogramming to cancer biology.
epigenetic memory; nuclear reprogramming; cancer; histone modifications; DNA methylation
Immune systems evolve as essential strategies to maintain homeostasis with the environment, prevent microbial assault and recycle damaged host tissues. The immune system is composed of two components, innate and adaptive immunity. The former is common to all animals while the latter consists of a vertebrate-specific system that relies on somatically derived lymphocytes and is associated with near limitless genetic diversity as well as long-term memory. Deuterostome invertebrates provide a view of immune repertoires in phyla that immediately predate the origins of vertebrates. Genomic studies in amphioxus, a cephalochordate, have revealed homologs of genes encoding most innate immune receptors found in vertebrates; however, many of the gene families have undergone dramatic expansions, greatly increasing the innate immune repertoire. In addition, domain-swapping accounts for the innovation of new predicted pathways of receptor function. In both amphioxus and Ciona, a urochordate, the VCBPs (variable region containing chitin-binding proteins), which consist of immunoglobulin V (variable) and chitin binding domains, mediate recognition through the V domains. The V domains of VCBPs in amphioxus exhibit high levels of allelic complexity that presumably relate to functional specificity. Various features of the amphioxus immune repertoire reflect novel selective pressures, which likely have resulted in innovative strategies. Functional genomic studies underscore the value of amphioxus as a model for studying innate immunity and may help reveal how unique relationships between innate immune receptors and both pathogens and symbionts factored in the evolution of adaptive immune systems.
innate immunity; Toll-like receptors; expanded immune repertoire; allelic complexity; gut immunity
The involvement of epigenetic processes in the origin and progression of cancer is now widely appreciated. Consequently, targeting the enzymatic machinery that controls the epigenetic regulation of the genome has emerged as an attractive new strategy for therapeutic intervention. The development of epigenetic drugs requires a detailed knowledge of the processes that govern chromatin regulation. Over the recent years, mass spectrometry (MS) has become an indispensable tool in epigenetics research. In this review, we will give an overview of the applications of MS-based proteomics in studying various aspects of chromatin biology. We will focus on the use of MS in the discovery and mapping of histone modifications and how novel proteomic approaches are being utilized to identify and study chromatin-associated proteins and multi-subunit complexes. Finally, we will discuss the application of proteomic methods in the diagnosis and prognosis of cancer based on epigenetic biomarkers and comment on their future impact on cancer epigenetics.
Mass spectrometry; quantitative proteomics; chromatin; histone modifications; epigenetic readers; cancer epigenetics
Carcinogenesis is thought to occur through a combination of mutational and epimutational events that disrupt key pathways regulating cellular growth and division. The DNA methylomes of cancer cells can exhibit two striking differences from normal cells; a global reduction of DNA methylation levels and the aberrant hypermethylation of some sequences, particularly CpG islands (CGIs). This aberrant hypermethylation is often invoked as a mechanism causing the transcriptional inactivation of tumour suppressor genes that directly drives the carcinogenic process. Here, we review our current understanding of this phenomenon, focusing on how global analysis of cancer methylomes indicates that most affected CGI genes are already silenced prior to aberrant hypermethylation during cancer development. We also discuss how genome-scale analyses of both normal and cancer cells have refined our understanding of the elusive mechanism(s) that may underpin aberrant CGI hypermethylation.
epigenetics; epigenomics; cancer; DNA methylation; CpG islands
The p53 transcription factor regulates the synthesis of mRNAs encoding proteins involved in diverse cellular stress responses such as cell-cycle arrest, apoptosis, autophagy and senescence. In this review, we discuss how these mRNAs are concurrently regulated at the post-transcriptional level by microRNAs (miRNAs) and RNA-binding proteins (RBPs), which consequently modify the p53 transcriptional program in a cell type- and stimulus-specific manner. We also discuss the action of specific miRNAs and RBPs that are direct transcriptional targets of p53 and how they act coordinately with protein-coding p53 target genes to orchestrate p53-dependent cellular responses.
p53; post-transcriptional regulation; RNA-binding proteins; miRNA
The regulation of mRNA translation is a major checkpoint in the flux of information from the transcriptome to the proteome. Critical for translational control are the trans-acting factors, RNA-binding proteins (RBPs) and small RNAs that bind to the mRNA and modify its translatability. This review summarizes the mechanisms by which RBPs regulate mRNA translation, with special focus on those binding to the 3′-untranslated region. It also discusses how recent high-throughput technologies are revealing exquisite layers of complexity and are helping to untangle translational regulation at a genome-wide scale.
RNA-binding protein; translation; UTR; RNP; CLIP; ribosome profiling
The mitogen-activated protein kinase kinases (the MAPK/ERK kinases; MKKs or MEKs) and their downstream substrates, the extracellular-regulated kinases have been intensively studied for their roles in development and disease. Until recently, it had been assumed any mutation affecting their function would have lethal consequences. However, the identification of MEK1 and MEK2 mutations in developmental syndromes as well as chemotherapy-resistant tumors, and the discovery of genomic variants in MEK1 and MEK2 have led to the realization the extent of genomic variation associated with MEKs is much greater than had been appreciated. In this review, we will discuss these recent advances, relating them to what is currently understood about the structure and function of MEKs, and describe how they change our understanding of the role of MEKs in development and disease.
MEK; MAPK; ERK; cardio-facial cutaneous syndrome; cancer; SNP
Piwi-interacting RNAs (piRNAs) and CRISPR RNAs (crRNAs) are two recently discovered classes of small noncoding RNA that are found in animals and prokaryotes, respectively. Both of these novel RNA species function as components of adaptive immune systems that protect their hosts from foreign nucleic acids—piRNAs repress transposable elements in animal germlines, whereas crRNAs protect their bacterial hosts from phage and plasmids. The piRNA and CRISPR systems are nonhomologous but rather have independently evolved into logically similar defense mechanisms based on the specificity of targeting via nucleic acid base complementarity. Here we review what is known about the piRNA and CRISPR systems with a focus on comparing their evolutionary properties. In particular, we highlight the importance of several factors on the pattern of piRNA and CRISPR evolution, including the population genetic environment, the role of alternate defense systems and the mechanisms of acquisition of new piRNAs and CRISPRs.
piRNA; CRISPR; co-evolution; transposable elements; phage; plasmids