Cancer cells and immune cells modulate their metabolism according to specific needs during cancer progression and immune responses. The ability to measure cellular metabolic function in vivo would enable the evaluation of tumors and their response to therapy and also the effectiveness of cellular immune responses to cancer. Positron emission tomography (PET) is a highly sensitive clinical imaging modality that enables whole-body, quantitative measurements of tissue biochemical function. Here, we review work using PET probes for specific metabolic pathways to measure cell function in cancer and immunity. We focus on the use of probes for glycolysis and nucleoside salvage and then discuss the development of new metabolic probes that visualize distinct parameters of cell function during disease.
When Takahashi and Yamanaka first demonstrated that just four transcription factors could reprogram a fibroblast to a pluripotent stem cell, the first wave of data to emerge focused on how similar these induced pluripotent stem cells (iPSCs) were to embryo-derived pluripotent stem cells (ESCs) . The next wave of data focused on determining the degree of difference between iPSCs and ESCs . Now the focus is on tweaking the process to generate iPSCs that are more similar to ESCs [3, 4]. Because transcription factor based reprogramming allows for nearly any type of cell to be created from any donor cell, there is obviously enormous interest in this technique as a tool for both basic developmental biology and for clinical applications. In this review, I will attempt to summarize the data that serve to distinguish these types of pluripotent stem cells and speculate on any ramifications of the differences.
Reprogramming of a somatic nucleus to an induced pluripotent state can be achieved in vitro through ectopic expression of Oct4 (Pou5f1), Sox2, Klf4 and c-Myc. While the ability of these factors to regulate transcription in a pluripotent context has been studied extensively, their ability to interact with and remodel a somatic genome remains underexplored. Several recent studies have begun to provide mechanistic insights that will eventually lead to a more rational design and improved understanding of nuclear reprogramming.
Pluripotency is a property that early embryonic cells possess over a considerable developmental time span. Accordingly, pluripotent cell lines can be established from the pre-implantation or post-implantation mouse embryo as embryonic stem (ES) or epiblast stem (EpiSC) cell lines, respectively. Maintenance of the pluripotent phenotype depends on the function of specific transcription factors (TFs) operating within a pluripotency gene regulatory network (PGRN). As cells move from an ES cell to an EpiSC state, the PGRN changes with expression of some TFs reduced (e.g. Nanog) or eliminated (e.g. Esrrb). Re-expressing such TFs can move cells back to an earlier developmental identity and is being applied to attempt establishment of human cell lines with the properties of mouse ES cells.
Long non-coding RNAs (ncRNAs) are emerging as important regulatory factors in mammalian genomics. A number of reports within the last 2 years have identified thousands of actively expressed long ncRNA transcripts with distinct properties. The long ncRNAs show differential expression patterns and regulation in a w ide variety of cells and tissues, adding significant complexity to the understanding of their biological role. Furthermore, genome-wide studies of transcriptional enhancers based on chromatin modifications and enhancer binding proteins have led to the identification of putative enhancers and provided insight into their tissue-specific regulation of gene expression. In an exciting turn of events, new evidence is indicating that long ncRNAs are associated with enhancer regions and that such non-coding transcription correlate with the increased activity of the neighboring genes. Moreover, additional experiments suggest that enhancer-function can be mediated through a transcribed long ncRNA and that this might be a common function for long ncRNAs. Here, we review recent advances made both in the genome-wide characterization of enhancers and in the identification of new classes of long ncRNAs, and discuss the functional overlap of these two classes of regulatory elements.
The advent of high-throughput technologies including deep-sequencing and protein mass spectrometry is facilitating the acquisition of large and precise data sets towards the definition of post-transcriptional regulatory networks. While early studies that investigated specific RNA-protein interactions in isolation laid the foundation for our understanding of the existence of molecular machines to assemble and process RNAs, there is a more recent appreciation of the importance of individual RNA-protein interactions that contribute to post-transcriptional gene regulation. The multitude of RNA-binding proteins (RBPs) and their many RNA targets has only been captured experimentally in recent times. In this review, we will examine current multidisciplinary approaches towards elucidating RNA-protein networks and their regulation.
The lin-4/miR-125 and let-7 microRNAs are at the heart of the heterochronic pathway, which controls temporal cell fate determination during Caenorhabditis elegans development. These small temporal RNAs are clustered along with a third microRNA, miR-100, in the genomes of most animals. Their conserved temporal and neural expression profile suggests a general role in cell fate determination during nervous system differentiation. By triggering consecutive differentiation programs, these microRNAs likely help to determine birth-order dependent temporal identity and thereby contribute to neural stem cell multipotency.
microRNAs (miRNAs) and small interfering RNAs (siRNAs), which constitute two major classes of endogenous small RNAs in plants, impact a multitude of developmental and physiological processes by imparting sequence specificity to gene and genome regulation. Although lacking the third major class of small RNAs found in animals, Piwi-interacting RNAs (piRNAs), plants have expanded their repertoire of endogenous siRNAs, some of which fulfill similar molecular and developmental functions as piRNAs in animals. Research on plant miRNAs and siRNAs has contributed invaluable insights into small RNA biology, thanks to the highly conserved molecular logic behind the biogenesis and actions of small RNAs. Here, I review progress in the plant small RNA field in the past two years, with an emphasis on recent findings related to plant development. I do not recount the numerous developmental processes regulated by small RNAs; instead, I focus on major principles that have been derived from recent studies and draw parallels, when applicable, between plants and animals.
argonaute; miRNA; siRNAs; piRNAs; cell-to-cell movement; germ line
Epithelial cells of the digestive tracts of most animals are short-lived, and are constantly replenished by the progeny of long-lived, resident intestinal stem cells. Proper regulation of intestinal stem cell maintenance, proliferation and differentiation is critical for maintaining gut homeostasis. Here we review recent genetic studies of stem cell-mediated homeostatic growth in the Drosophila midgut and the mouse small intestine, highlighting similarities and differences in the mechanisms that control stem cell proliferation and differentiation.
The cell surface receptor Notch contributes to the development of nearly every tissue in most metazoans by controlling the fates and differentiation of cells. Recent results have now established that Notch also regulates the connectivity of the nervous system, and does so at a variety of levels, including specification of neuronal identity, division, survival and migration, as well as axon guidance, morphogenesis of dendritic arbors and weighting of synapse strength. To these ends, Notch engages at least two signal transduction pathways, one that controls nuclear gene expression and another that directly targets the cytoskeleton. Coordinating the many functions of Notch to produce neural structure is thus a pivotal aspect of building and maintaining the nervous system.
Piwi proteins, together with their bound Piwi-interacting RNAs, constitute an evolutionarily conserved, germline-specific innate immune system. The piRNA pathway is one of the key mechanisms for silencing transposable elements in the germline, thereby preserving genome integrity between generations. Recent work from several groups has significantly advanced our understanding of how piRNAs arise from discrete genomic loci, termed piRNA clusters, and how these Piwi-piRNA complexes enforce transposon silencing. Here, we discuss these recent findings, as well as highlight some aspects of piRNA biology that continue to escape our understanding.
Recent advances in microscopy and genomic techniques have provided new insight into spatial chromatin organization inside of the nucleus. In particular, chromosome conformation capture data has highlighted the relevance of polymer physics for high-order chromatin organization. In this context, we review basic polymer states, discuss how an appropriate polymer model can be determined from experimental data, and examine the success and limitations of various polymer models of high-order interphase chromatin organization. By taking into account topological constraints acting on the chromatin fiber, recently-developed polymer models of interphase chromatin can reproduce the observed scaling of distances between genomic loci, chromosomal territories, and probabilities of contacts between loci measured by chromosome conformation capture methods. Polymer models provide a framework for the interpretation of experimental data as ensembles of conformations rather than collections of loops, and will be crucial for untangling functional implications of chromosomal organization.
Pathways linking oncogenic mutations to increased proliferative or migratory capacity are poorly characterized, yet provide potential targets for therapeutic intervention. As tyrosine phosphorylation signaling networks are known to mediate proliferation and migration, and frequently go awry in cancers, a comprehensive understanding of these networks in normal and diseased states is warranted. To this end, recent advances in mass spectrometry, protein microarrays, and computational algorithms provide insight into various aspects of the network including phosphotyrosine identification, analysis of kinase/phosphatase-substrates, and phosphorylation mediated protein-protein interactions. Here we detail technological advances underlying these systems level approaches and give examples of their application. By combining multiple approaches, it is now possible to quantify changes in the phosphotyrosine signaling network with various oncogenic mutations, thereby unveiling novel therapeutic targets.
Systems biology; tyrosine phosphorylation; cellular signaling; mass spectrometry
Axonal growth cones (GCs) steer in response to extrinsic cues using mechanisms that include local protein synthesis. This adaptive form of gene regulation occurs with spatial precision and depends on subcellular mRNA localisation. Recent genome-wide studies have shown unexpectedly complex and dynamically changing mRNA repertoires in growing axons and GCs. Axonal targeting of some transcripts seems to be highly selective and involves sequence diversity in non-coding regions generated by transcriptional and/or posttranscriptional mechanisms. New evidence reports direct coupling of a guidance receptor to the protein synthesis machinery and other findings demonstrate that some guidance cues can repress translation. The recent findings shed further light on the exquisitely regulated process that enables distant cellular compartments to respond to local stimuli.
An intriguing set of neurodegenerative disease are the nine disorders caused by the expansion of a unstable trinucleotide CAG repeat where the repeat is located within the coding of the affected gene, i.e. the polyglutamine (polyQ) diseases. A gain-of-function mechanism for toxicity in polyQ diseases is widely thought to have a major role in pathogenesis. Yet, the specific nature of this gain-of-function is a matter of considerable discussion. The basic issue concerns whether toxicity stems from the native or normal function of the affected protein versus a novel function induced by polyQ expansion. For at least three of the polyQ disease considerable evidence is accumulating that pathology is mediated by a polyQ-induced exaggeration of a native function of the host protein.
Copy number variants (CNVs) are widely distributed throughout the human genome, where they contribute to genetic variation and phenotypic diversity. De novo CNVs are also a major cause of numerous genetic and developmental disorders. However, unlike many other types of mutations, little is known about the genetic and environmental risk factors for new and deleterious CNVs. DNA replication errors have been implicated in the generation of a major class of CNVs, the nonrecurrent CNVs. We have found that agents that perturb normal replication and create conditions of replication stress, including hydroxyurea and aphidicolin, are potent inducers of nonrecurrent CNVs in cultured human cells. These findings have broad implications for identifying CNV risk factors and for hydroxyurea-related therapies in humans.
Mobile DNAs, also known as transposons or “jumping genes”, are widespread in nature and comprise an estimated 45% of the human genome. Transposons are divided into two general classes based on their transposition intermediate (DNA or RNA). Only one subclass, non-LTR retrotransposons, is currently active in humans as indicated by 96 disease-causing insertions. These autonomous Long INterspersed Element-1s (LINE-1s or L1s) are capable of retrotransposing not only a copy of their own RNA but also other RNAs (Alu, SINE-VNTR-Alu (SVA), U6) in trans to new genomic locations through an element encoded reverse transcriptase. L1 can also retrotranspose cellular mRNAs, resulting in processed pseudogene formation. Here, we highlight recent reports that update our understanding of human L1 retrotransposition and their role in disease. Finally we discuss studies that provide insights into the past and current activity of these retrotransposons, and shed light on not just when, but where, retrotransposition occurs and its part in genetic variation.
Repetitive DNA sequences constitute 30% of the human genome, and are often sites of genomic rearrangement. Recently, it has been found that several constitutional translocations, especially those that involve chromosome 22, take place utilizing palindromic sequences on 22q11 and on the partner chromosome. Analysis of translocation junction fragments shows that the breakpoints of such palindrome-mediated translocations are localized at the center of palindromic AT-rich repeats (PATRRs). The presence of PATRRs at the breakpoints, indicates a palindrome-mediated mechanism involved in the generation of these constitutional translocations. Identification of these PATRR-mediated translocations suggests a universal pathway for gross chromosomal rearrangement in the human genome. De novo occurrences of PATRR-mediated translocations can be detected by PCR in normal sperm samples but not somatic cells. Polymorphisms of various PATRRs influence their propensity for adopting a secondary structure, which in turn affects de novo translocation frequency. We propose that the PATRRs form an unstable secondary structure, which leads to double-strand breaks at the center of the PATRR. The double-strand breaks appear to be followed by a non-homologous end-joining repair pathway, ultimately leading to the translocations. This review considers recent findings concerning the mechanism of meiosis-specific, PATRR-mediated translocations.
During the last two decades, the importance of human genome copy number variation (CNV) in disease has become widely recognized. However, much is not understood about underlying mechanisms. We show how, although model organism research guides molecular understanding, important insights are gained from study of the wealth of information available in the clinic. We describe progress in explaining nonallelic homologous recombination (NAHR), a major cause of copy number change occurring when control of allelic recombination fails, highlight the growing importance of replicative mechanisms to explain complex events, and describe progress in understanding extreme chromosome reorganization (chromothripsis). Both non-homologous end-joining and aberrant replication have significant roles in chromothripsis. As we study CNV, the processes underlying human genome evolution are revealed.
NAHR; FoSTeS; MMBIR; ectopic synapsis; PRDM9; triplication; chromothripsis
The last decade has witnessed an explosion in the identification of genes, mutations in which appear sufficient to cause clinical phenotypes in humans. This is especially true for disorders of ciliary dysfunction in which an excess of 50 causal loci are now known; this discovery was driven in part by an improved understanding of the protein composition of the cilium and the co-occurrence of clinical phenotypes associated with ciliary dysfunction. Despite this progress, the fundamental challenge of predicting phenotype and or clinical progression based on single locus information remains unsolved. Here, we explore how the combinatorial knowledge of allele quality and quantity, an improved understanding of the biological composition of the primary cilium, and the expanded appreciation of the subcellular roles of this organelle can be synthesized to generate improved models that can explain both causality but also variable penetrance and expressivity.
Fragile X syndrome (FXS) is a trinucleotide repeat disorder caused by a CGG repeat expansion in FMR1, and loss of its protein product FMRP. Recent studies have provided increased support for the role of FMRP in translational repression via ribosomal stalling and the microRNA pathway. In neurons, particular focus has been placed on identifying the signaling pathways such as PI3K and mTOR downstream of group 1 metabotropic glutamate receptors (mGluR1/5) that regulate FMRP. New evidence also suggests that loss of FMRP causes presynaptic dysfunction and abnormal adult neurogenesis. In addition, studies on FXS stem cells especially induced pluripotent stem (iPS) cells and new sequencing efforts hold out promise for deeper understanding of the silencing process and mutation spectrum of FMR1.
fragile X syndrome; intellectual disability (ID); translational regulation; ribosomal stalling; microRNAs; long term depression (LTD); group 1 metabotropic glutamate receptors; presynapse; adult neurogenesis; human embryonic stem cell (hESC); induced pluripotent stem cell (iPS); missense mutation
Prion diseases are transmissible, fatal neurodegenerative diseases that include scrapie and bovine spongiform encephalopathy (BSE) in animals and Creutzfeldt–Jakob disease (CJD) in human. The prion protein gene (PRNP) is the major genetic determinant of susceptibility, however, several studies now suggest that other genes are also important. Two recent genome wide association studies in human have identified four new loci of interest: ZBTB38-RASA2 in UK CJD cases and MTMR7 and NPAS2 in variant CJD. Complementary studies in mouse have used complex crosses to identify new modifiers such as Cpne8 and provided supporting evidence for previously implicated genes (Rarb and Stmn2). Expression profiling has identified new candidates, including Hspa13, which reduces incubation time in a transgenic model.
Nuclear transfer (NT) remains the most effective method to reprogram somatic cells to totipotency. Somatic cell nuclear transfer (SCNT) efficiency however remains low, but recurrent problems occurring in partially reprogrammed cloned embryos have recently been identified and some remedied. In particular, the trophectoderm has been identified as a lineage whose reprogramming success has a large influence on SCNT embryo development. Several interspecific hybrid and cybrid reprogramming systems have been developed as they offer various technical advantages and potential applications, and together with SCNT, they have led to the identification of a series of reprogramming events and responsible reprogramming factors. Interspecific incompatibilities hinder full exploitation of cross-species reprogramming systems, yet recent findings suggest that these may not constitute insurmountable obstacles.
Nuclear reprogramming; Somatic cell nuclear transfer (SCNT); Interspecies SCNT (iSCNT); Hybrid; Nucleocytoplasmic hybrid (Cybrid); Nucleocytoplasmic incompatibility; Microcell-mediated chromosome transfer (MMCT); Interspecies intra-cytoplasmic sperm injection (iICSI); Cell fusion
The importance of mechanical signals during embryogenesis and development, through both intercellular and extracellular signals, are coming into focus. It is widely hypothesized that physical forces help to guide the shape, cellular differentiation and the patterning of tissues. To test these ideas many classical engineering principles and imaging technologies are being adapted. Recent advances in microscopy, mechanical testing and genetic and pharmacological techniques, alongside computational models are helping to dissect the activity of mechanical signals in development at the cellular and molecular level. These inroads are permitting the study of mechanical changes in tissue structure and stiffness, and will provide deeper insights into the role of mechanics in both developmental biology and disease.
deformation; cell shape change; tissue and cellular mechanics; stress; traction; modulus; elasticity; viscoelasticity
Genomic imprinting is an epigenetic process resulting in the monoallelic parent-of-origin-specific expression of a subset of genes in the mammalian genome. The parental alleles are differentially marked by DNA methylation during gametogenesis when the genomes are in separate compartments. How methylation machinery recognizes and differentially modifies these imprinted regions in germ cells remains a key question in the field. While studies have focused on determining a sequence signature that alone could distinguish imprinted regions from the rest of the genome, recent reports do not support such a hypothesis. Rather, it is becoming clear that features such as transcription, histone modifications and higher order chromatin are employed either individually or in combination to set up parental imprints.