Stochastic gene expression causes phenotypic heterogeneity in a population of genetically identical bacterial cells. Such non-genetic heterogeneity can have important consequences for the population fitness, and therefore cells implement regulation strategies to either suppress or exploit such heterogeneity to adapt to their circumstances. By employing time-lapse microscopy of single cells, the fluctuation dynamics of gene expression may be analysed, and their regulatory mechanisms thus deciphered. However, a careful consideration of the experimental design and data-analysis is needed to produce useful data for deriving meaningful insights from them. In the present paper, the individual steps and challenges involved in a time-lapse experiment are discussed, and a rigorous framework for designing, performing, and extracting single-cell gene expression dynamics data from such experiments is outlined.

In this review I focus on the role of splicing in long non-coding RNA (lncRNA) life. First, I summarize differences between the splicing efficiency of protein-coding genes and lncRNAs and discuss why non-coding RNAs are spliced less efficiently. In the second half of the review, I speculate why splice sites are the most conserved sequences in lncRNAs and what additional roles could splicing play in lncRNA metabolism. I discuss the hypothesis that the splicing machinery can, besides its dominant role in intron removal and exon joining, protect cells from undesired transcripts.

Recent efforts on the characterization of long non-coding RNAs (lncRNAs) revealed their functional roles in modulating diverse cellular processes. These include pluripotency maintenance, lineage commitment, carcinogenesis, and pathogenesis of various diseases. By interacting with DNA, RNA and protein, lncRNAs mediate multifaceted mechanisms to regulate transcription, RNA processing, RNA interference and translation. Of more than 173000 discovered lncRNAs, the majority remain functionally unknown. The cell type-specific expression and localization of the lncRNA also suggest potential distinct functions of lncRNAs across different cell types. This highlights the niche of identifying functional lncRNAs in different biological processes and diseases through high-throughput (HTP) screening. This review summarizes the current work performed and perspectives on HTP screening of functional lncRNAs where different technologies, platforms, cellular responses and the downstream analyses are discussed. We hope to provide a better picture in applying different technologies to facilitate functional annotation of lncRNA efficiently.

The genomes of complex eukaryotes largely contain non-protein-coding DNA, which is pervasively transcribed into a plethora of non-coding RNAs (ncRNAs). The functional importance of many of these ncRNAs has been investigated in the last two decades, revealing their crucial and multifaceted roles in chromatin regulation. A common mode of action of ncRNAs is the recruitment of chromatin modifiers to specific regions in the genome. Whereas many ncRNA–protein interactions have been characterised in detail, binding of ncRNAs to their DNA target sites is much less understood. Recently developed RNA-centric methods have mapped the genome-wide distribution of ncRNAs, however, how ncRNAs achieve locus-specificity remains mainly unresolved. In terms of direct RNA–DNA interactions, two kinds of triple-stranded structures can be formed: R-loops consisting of an RNA:DNA hybrid and a looped out DNA strand, and RNA:DNA triple helices (triplexes), in which the RNA binds to the major groove of the DNA double helix by sequence-specific Hoogsteen base pairing. In this essay, we will review the current knowledge about RNA:DNA triplexes, summarising triplex formation rules, detection methods, and ncRNAs reported to engage in triplexes. While the functional characterisation of RNA:DNA triplexes is still anecdotal, recent advances in high-throughput and computational analyses indicate their widespread distribution in the genome. Thus, we are witnessing a paradigm shift in the appreciation of RNA:DNA triplexes, away from exotic structures towards a prominent mode of ncRNA–chromatin interactions.

Paraspeckles are a type of subnuclear bodies built on the long noncoding RNA NEAT1 (nuclear paraspeckle assembly transcript 1, also known as MEN-ε/β or VINC-1) . Paraspeckles are involved in many physiological processes including cellular stress responses, cell differentiation, corpus luteum formation and cancer progression. Recently, ultra-resolution microscopy coupled with multicolor-labeling of paraspeckle components (the NEAT1 RNA and paraspeckle proteins) revealed the exquisite details of paraspeckle structure and function. NEAT1 transcripts are radially arranged to form a core–shell spheroidal structure, while paraspeckle proteins (PSPs) localize within different layers. Functional dissection of NEAT1 shows that the subdomains of NEAT1_2 are important for RNA stability, isoform switching and paraspeckle assembly via a liquid–liquid phase separation (LLPS) mechanism. We review recent progress on structure and organization of paraspeckles as well as how paraspeckles spatiotemporally control gene regulation through sequestration of diverse proteins and RNAs in cells.

In eukaryotes, RNAs newly synthesized by RNA polymerase II (RNAPII) undergo several processing steps prior to transport to the cytoplasm. It has long been known that RNAs with defects in processing or export are removed in the nucleus. Recent studies revealed that RNAs without apparent defects are also subjected to nuclear degradation, indicating that nuclear RNA fate is determined in a more complex and dynamic way than previously thought. Nuclear RNA sorting directly determines the quality and quantity of RNA pools for future translation and thus is of significant importance. In this essay, we will summarize recent studies on this topic, mainly focusing on findings in mammalian system, and discuss about important remaining questions and possible biological relevance for nuclear RNA fate determination.

In plants, post-transcriptional gene silencing (PTGS) tightly regulates development, maintains genome stability and protects plant against foreign genes. PTGS can be triggered by virus infection, transgene, and endogenous transcript, thus commonly serves as an RNA-based immune mechanism. Accordingly, based on the initiating factors, PTGS can be divided into viral-PTGS, transgene-PTGS, and endo-gene-PTGS. Unlike the intensely expressed invading transgenes and viral genes that frequently undergo PTGS, most endogenous genes do not trigger PTGS, except for a few that can produce endogenous small RNAs (sRNAs), including microRNA (miRNA) and small interfering RNA (siRNA). Different lengths of miRNA and siRNA, mainly 21-, 22- or 24-nucleotides (nt) exert diverse functions, ranging from target mRNA degradation, translational inhibition, or DNA methylation and chromatin modifications. The abundant 21-nt miRNA or siRNA, processed by RNase-III enzyme DICER-LIKE 1 (DCL1) and DCL4, respectively, have been well studied in the PTGS pathways. By contrast, the scarceness of endogenous 22-nt sRNAs that are primarily processed by DCL2 limits their research, although a few encouraging studies have been reported recently. Therefore, we review here our current understanding of diverse PTGS pathways triggered by a variety of sRNAs and summarize the distinct features of the 22-nt sRNA mediated PTGS.

Transcription factors (TFs) are well-established key factors orchestrating gene transcription, and RNA-binding proteins (RBPs) are mainly thought to participate in post-transcriptional control of gene. In fact, these two steps are functionally coupled, offering a possibility for reciprocal communications between transcription and regulatory RNAs and RBPs. Recently, a series of exploratory studies, utilizing functional genomic strategies, have revealed that RBPs are prevalently involved in transcription control genome-wide through their interactions with chromatin. Here, we present a refined census of RBPs to grope for such an emerging role and discuss the global view of RBP–chromatin interactions and their functional diversities in transcription regulation.

microRNAs (miRNAs) play essential roles in mouse embryonic stem cells (ESCs) and early embryo development. The exact mechanism by which miRNAs regulate cell fate transition during embryo development is still not clear. Recent studies have identified and captured various pluripotent stem cells (PSCs) that share similar characteristics with cells from different stages of pre- and post-implantation embryos. These PSCs provide valuable models to understand miRNA functions in early mammalian development. In this short review, we will summarize recent work towards understanding the function and mechanism of miRNAs in regulating the transition or conversion between different pluripotent states. In addition, we will highlight unresolved questions and key future directions related to miRNAs in pluripotent state transition. Studies in these areas will further our understanding of miRNA functions in early embryo development, and may lead to practical means to control human PSCs for clinical applications in regenerative medicine.

Enhancers are noncoding DNA elements that are present upstream or downstream of a gene to control its spatial and temporal expression. Specific histone modifications, such as monomethylation on histone H3 lysine 4 (H3K4me1) and H3K27ac, have been widely used to assign enhancer regions in mammalian genomes. In recent years, emerging evidence suggests that active enhancers are bidirectionally transcribed to produce enhancer RNAs (eRNAs). This finding not only adds a new reliable feature to define enhancers but also raises a fundamental question of how eRNAs function to activate transcription. Although some believe that eRNAs are merely transcriptional byproducts, many studies have demonstrated that eRNAs execute crucial tasks in regulating chromatin conformation and transcription activation. In this review, we summarize the current understanding of eRNAs from their biogenesis, functions, and regulation to their pathological significance. Additionally, we discuss the challenges and possible mechanisms of eRNAs in regulated transcription.

Precursor RNAs undergo extensive processing to become mature RNAs. RNA transcripts are subjected to 5′ capping, 3′-end processing, splicing, and modification; they also form dynamic secondary structures during co-transcriptional and post-transcriptional processing. Like coding RNAs, non-coding RNAs (ncRNAs) undergo extensive processing. For example, secondary small interfering RNA (siRNA) transcripts undergo RNA processing, followed by further cleavage to become mature siRNAs. Transcriptome studies have revealed roles for co-transcriptional and post-transcriptional RNA processing in the regulation of gene expression and the coordination of plant development and plant–environment interactions. In this review, we present the latest progress on RNA processing in gene expression and discuss phased siRNAs (phasiRNAs), a kind of germ cell-specific secondary small RNA (sRNA), focusing on their functions in plant development and environmental responses.

Amyotrophic lateral sclerosis (ALS) is a rapidly disabling and fatal neurodegenerative disease. Due to insufficient disease-modifying treatments, there is an unmet and urgent need for elucidating disease mechanisms that occur early and represent common triggers in both familial and sporadic ALS. Emerging evidence suggests that impaired DNA damage response contributes to age-related somatic accumulation of genomic instability and can trigger or accelerate ALS pathological manifestations. In this review, we summarize and discuss recent studies indicating a direct link between DNA damage response and ALS. Further mechanistic understanding of the role genomic instability is playing in ALS disease pathophysiology will be critical for discovering new therapeutic avenues.

The mechanisms by which RNA acts in the DNA damage response (DDR), specifically in the repair of DNA double-strand breaks (DSBs), are emerging as multifaceted and complex. Different RNA species, including but not limited to; microRNA (miRNA), long non-coding RNA (lncRNA), RNA:DNA hybrid structures, the recently identified damage-induced lncRNA (dilncRNA), damage-responsive transcripts (DARTs), and DNA damage-dependent small RNAs (DDRNAs), have been shown to play integral roles in the DSB response. The diverse properties of these RNAs, such as sequence, structure, and binding partners, enable them to fulfil a variety of functions in different cellular contexts. Additionally, RNA can be modified post-transcriptionally, a process which is regulated in response to cellular stressors such as DNA damage. Many of these mechanisms are not yet understood and the literature contradictory, reflecting the complexity and expansive nature of the roles of RNA in the DDR. However, it is clear that RNA is pivotal in ensuring the maintenance of genome integrity. In this review, we will discuss and summarise recent evidence which highlights the roles of these various RNAs in preserving genomic integrity, with a particular focus on the emerging role of RNA in the DSB repair response.

DNA methylation is an epigenetic mark involved in regulating genome function and is critical for normal development in mammals. It has been observed that the developmental environment can lead to permanent changes in gene expression and DNA methylation, at least at ‘metastable epialleles’. These are defined as regions of the genome that show a variable epigenetic state that is established early in development and maintained through subsequent cell divisions. However, the majority of the known genome does not behave in this manner. Here, we use the developmental origins of adult disease hypothesis to understand environmental epigenomics. Some challenges to studying how DNA methylation is influenced by the environment include identifying DNA methylation changes associated with an environmental exposure in tissues with a complex cellular composition and at genomic regions for which DNA methylation is dynamically regulated in a cell-type specific manner. We also offer a perspective of how emerging technologies may be useful for dissecting the functional contribution of exposure-associated epigenetic changes and highlight recent evidence that suggests that genomic regions that are absent from genome assemblies may be unappreciated hotspots for environmental modulation of the epigenetic state.

Monoallelic gene expression occurs in diploid cells when only one of the two alleles of a gene is active. There are three main classes of genes that display monoallelic expression in mammalian genomes: (1) imprinted genes that are monoallelically expressed in a parent-of-origin dependent manner; (2) X-linked genes that undergo random X-chromosome inactivation in female cells; (3) random monoallelically expressed single and clustered genes located on autosomes. The heritability of monoallelic expression patterns during cell divisions implies that epigenetic mechanisms are involved in the cellular memory of these expression states. Among these, methylation of CpG sites on DNA is one of the best described modification to explain somatic inheritance. Here, we discuss the relevance of DNA methylation for the establishment and maintenance of monoallelic expression patterns among these three groups of genes, and how this is intrinsically linked to development and cellular states.

Epigenetic information in the mammalian oocyte has the potential to be transmitted to the next generation and influence gene expression; this occurs naturally in the case of imprinted genes. Therefore, it is important to understand how epigenetic information is patterned during oocyte development and growth. Here, we review the current state of knowledge of de novo DNA methylation mechanisms in the oocyte: how a distinctive gene-body methylation pattern is created, and the extent to which the DNA methylation machinery reads chromatin states. Recent epigenomic studies building on advances in ultra-low input chromatin profiling methods, coupled with genetic studies, have started to allow a detailed interrogation of the interplay between DNA methylation establishment and chromatin states; however, a full mechanistic description awaits.

Cytosine methylation is a DNA modification that is critical for vertebrate development and provides a plastic yet stable information module in addition to the DNA code. DNA methylation memory establishment, maintenance and erasure is carefully balanced by molecular machinery highly conserved among vertebrates. In mammals, extensive erasure of epigenetic marks, including 5-methylcytosine (5mC), is a hallmark of early embryo and germline development. Conversely, global cytosine methylation patterns are preserved in at least some non-mammalian vertebrates over comparable developmental windows. The evolutionary mechanisms which drove this divergence are unknown, nevertheless a direct consequence of retaining epigenetic memory in the form of 5mC is the enhanced potential for transgenerational epigenetic inheritance (TEI). Given that DNA methylation dynamics remains underexplored in most vertebrate lineages, the extent of information transferred to offspring by epigenetic modification might be underestimated.

Dynamic binding of transcription factors (TFs) to regulatory elements controls transcriptional states throughout organism development. Epigenetics modifications, such as DNA methylation mostly within cytosine-guanine dinucleotides (CpGs), have the potential to modulate TF binding to DNA. Although DNA methylation has long been thought to repress TF binding, a more recent model proposes that TF binding can also inhibit DNA methylation. Here, we review the possible scenarios by which DNA methylation and TF binding affect each other. Further in vivo experiments will be required to generalize these models.

DNA methylation is an essential DNA modification that plays a crucial role in genome regulation during differentiation and development, and is disrupted in a range of disease states. The recent development of CRISPR/catalytically dead CRISPR/Cas9 (dCas9)-based targeted DNA methylation editing tools has enabled new insights into the roles and functional relevance of this modification, including its importance at regulatory regions and the role of aberrant methylation in various diseases. However, while these tools are advancing our ability to understand and manipulate this regulatory layer of the genome, they still possess a variety of limitations in efficacy, implementation, and targeting specificity. Effective targeted DNA methylation editing will continue to advance our fundamental understanding of the role of this modification in different genomic and cellular contexts, and further improvements may enable more accurate disease modeling and possible future treatments. In this review, we discuss strategies, considerations, and future directions for targeted DNA methylation editing.

DNA methylation is an epigenetic modification essential for normal mammalian development. Initially associated with gene silencing, more diverse roles for DNA methylation in the regulation of gene expression patterns are increasingly being recognized. Some of these insights come from studying the function of genes that are mutated in human diseases characterized by abnormal DNA methylation landscapes. The first disorder to be associated with congenital defects in DNA methylation was Immunodeficiency, Centromeric instability, Facial anomalies syndrome (ICF). The hallmark of this syndrome is hypomethylation of pericentromeric satellite repeats, with mutations in four genes: DNMT3B, ZBTB24, CDCA7 and HELLS , being linked to the disease. Here, we discuss recent progress in understanding the molecular interactions between these genes and consider current evidence for how aberrant DNA methylation may contribute to the abnormal phenotype present in ICF syndrome patients.

Nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), carry genetic information which is read in cells to make the RNA and proteins by which living things function. The well-known structure of the DNA double helix allows this information to be copied and passed on to the next generation. In this article we summarise the structure and function of nucleic acids. The article includes a historical perspective and summarises some of the early work which led to our understanding of this important molecule and how it functions; many of these pioneering scientists were awarded Nobel Prizes for their work. We explain the structure of the DNA molecule, how it is packaged into chromosomes and how it is replicated prior to cell division. We look at how the concept of the gene has developed since the term was first coined and how DNA is copied into RNA (transcription) and translated into protein (translation).

Cell migration is a multifactorial/multistep process that requires the concerted action of growth and transcriptional factors, motor proteins, extracellular matrix remodeling and proteases. In this review, we focus on the role of transcription factors modulating Epithelial-to-Mesenchymal Transition (EMT-TFs), a fundamental process supporting both physiological and pathological cell migration. These EMT-TFs (Snail1/2, Twist1/2 and Zeb1/2) are labile proteins which should be stabilized to initiate EMT and provide full migratory and invasive properties. We present here a family of enzymes, the deubiquitinases (DUBs) which have a crucial role in counteracting polyubiquitination and proteasomal degradation of EMT-TFs after their induction by TGFβ, inflammatory cytokines and hypoxia. We also describe the DUBs promoting the stabilization of Smads, TGFβ receptors and other key proteins involved in transduction pathways controlling EMT.

Transposable elements dominate the mammalian genome, but their contribution to genetic and epigenetic regulation has been largely overlooked. This was in part due to technical limitations, which made the study of repetitive sequences at single copy resolution difficult. The advancement of next-generation sequencing assays in the last decade has greatly enhanced our understanding of transposable element function. In some instances, specific transposable elements are thought to have been co-opted into regulatory roles during both mouse and human development, while in disease such regulatory potential can contribute to malignancy. DNA methylation is arguably the best characterised regulator of transposable element activity. DNA methylation is associated with transposable element repression, and acts to limit their genotoxic potential. In specific developmental contexts, erasure of DNA methylation is associated with a burst of transposable element expression. Developmental regulation of DNA methylation enables transposon activation, ensuring their survival and propagation throughout the host genome, and also allows the host access to regulatory sequences encoded within the elements. Here I discuss DNA methylation at transposable elements, describing its function and dynamic regulation throughout murine and human development.

Cell migration is a fundamental biological process involved in tissue formation and homeostasis. The correct polarization of motile cells is critical to ensure directed movement, and is orchestrated by many intrinsic and extrinsic factors. Of these, the subcellular distribution of mRNAs and the consequent spatial control of translation are key modulators of cell polarity. mRNA transport is dependent on cis -regulatory elements within transcripts, which are recognized by trans -acting proteins that ensure the efficient delivery of certain messages to the leading edge of migrating cells. At their destination, translation of localized mRNAs then participates in regional cellular responses underlying cell motility. In this review, we summarize the key findings that established mRNA targetting as a critical driver of cell migration and how the characterization of polarized mRNAs in motile cells has been expanded from just a few species to hundreds of transcripts. We also describe the molecular control of mRNA trafficking, subsequent mechanisms of local protein synthesis and how these ultimately regulate cell polarity during migration.

The organization and regulation of genomic DNA as nuclear chromatin is necessary for proper DNA function inside living eukaryotic cells. While this has been extensively explored, no true consensus is currently reached regarding the exact mechanism of chromatin organization. The traditional view has assumed that the DNA is packaged into a hierarchy of structures inside the nucleus based on the regular 30-nm chromatin fiber. This is currently being challenged by the fluid-like model of the chromatin which views the chromatin as a dynamic structure based on the irregular 10-nm fiber. In this review, we focus on the recent progress in chromatin structure elucidation highlighting the paradigm shift in chromatin folding mechanism from the classical textbook perspective of the regularly folded chromatin to the more dynamic fluid-like perspective.

ATP-dependent chromatin remodelling enzymes play a fundamental role in determining how nucleosomes are organised, and render DNA sequences accessible to interacting proteins, thereby enabling precise regulation of eukaryotic genes. Remodelers conserved from yeast to humans are classified into four families based on the domains and motifs present in their ATPase subunits. Insights into overall assembly and the mode of interaction to the nucleosome by these different families of remodelers remained limited due to the complexity of obtaining structural information on these challenging samples. Electron microscopy and single-particle methods have made advancement and uncovered vital structural information on the number of remodelling complexes. In this article, we highlight some of the recent structural work that advanced our understanding on the mechanisms and biological functions of these ATP-dependent remodelling machines.

Nucleosomes cover eukaryotic genomes like beads on a string and play a central role in regulating genome function. Isolated strings of nucleosomes have the potential to compact and form higher order chromatin structures, such as the well-characterized 30-nm fiber. However, despite tremendous advances in observing chromatin fibers in situ it has not been possible to confirm that regularly ordered fibers represent a prevalent structural level in the folding of chromosomes. Instead, it appears that folding at a larger scale than the nucleosome involves a variety of random structures with fractal characteristics. Nevertheless, recent progress provides evidence for the existence of structural motifs in chromatin fibers, potentially localized to strategic sites in the genome. Here we review the current understanding of chromatin fiber folding and the emerging roles that oligonucleosomal motifs play in the regulation of genome function.

Packaging the long and fragile genomes of eukaryotic species into nucleosomes is all well and good, but how do cells gain access to the DNA again after it has been bundled away? The solution, in every species from yeast to man, is to post-translationally modify histones, altering their chemical properties to either relax the chromatin, label it for remodelling or make it more compact still. Histones are subject to a myriad of modifications: acetylation, methylation, phosphorylation, ubiquitination etc. This review focuses on histone acylations, a diverse group of modifications which occur on the ε-amino group of Lysine residues and includes the well-characterised Lysine acetylation. Over the last 50 years, histone acetylation has been extensively characterised, with the discovery of histone acetyltransferases (HATs) and histone deacetylases (HDACs), and global mapping experiments, revealing an association of hyperacetylated histones with accessible, transcriptionally active chromatin. More recently, there has been an explosion in the number of unique short chain ‘acylations’ identified by MS, including: propionylation, butyrylation, crotonylation, succinylation, malonylation and 2-hydroxyisobutyrylation. These novel modifications add a range of chemical environments to histones, and similar to acetylation, appear to accumulate at transcriptional start sites and correlate with gene activity.

The eukaryotic genome is regulated in the context of chromatin. Specialized histones, known as histone variants, incorporate into chromatin to replace their canonical counterparts and represent an important layer of regulation to diversify the structural characteristics and functional outputs of chromatin. MacroH2A is an unusual histone variant with a bulky C-terminal non-histone domain that distinguishes it from all other histones. It is a critical player in stabilizing differentiated cell identity by posing as a barrier to somatic cell reprogramming toward pluripotency and acts as a tumor suppressor in a wide range of cancers. MacroH2A histones are generally regarded as repressive variants that are enriched at the inactive X chromosome (Xi) and broad domains across autosomal chromatin. Recent studies have shed light on to how macroH2A influences transcriptional outputs within distinct genomic contexts and revealed new intriguing molecular functions of macroH2A variants beyond transcriptional regulation. Furthermore, the mechanisms of its mysterious chromatin deposition are beginning to be unraveled, facilitating our understanding of its complex regulation of genome function.

Orchestrating vertebrate genomes require a complex interplay between the linear composition of the genome and its 3D organization inside the nucleus. This requires the function of specialized proteins, able to tune various aspects of genome organization and gene regulation. The CCCTC-binding factor (CTCF) is a DNA binding factor capable of regulating not only the 3D genome organization, but also key aspects of gene expression, including transcription activation and repression, RNA splicing, and enhancer/promoter insulation. A growing body of evidence proposes that CTCF, together with cohesin contributes to DNA loop formation and 3D genome organization. CTCF binding sites are mutation hotspots in cancer, while mutations in CTCF itself lead to intellectual disabilities, emphasizing its importance in disease etiology. In this review we cover various aspects of CTCF function, revealing the polyvalence of this factor as a highly diversified tool for vertebrate genome organization and transcription regulation.

Chromatin comprises proteins, DNA and RNA, and its function is to condense and package the genome in a way that allows the necessary transactions such as transcription, replication and repair to occur in a highly organised and regulated manner. The packaging of chromatin is often thought of in a hierarchical fashion starting from the most basic unit of DNA packaging, the nucleosome, to the condensation of nucleosomal ‘beads on a string’ by linker histones to form the 30-nm fibre and eventually large chromatin domains. However, a picture of a more heterogeneous, dynamic and liquid-like assembly is emerging, in which intrinsically disordered proteins (IDPs) and proteins containing intrinsically disordered regions (IDRs) play a central role. Disorder features at all levels of chromatin organisation, from the histone tails, which are sites of extensive post-translational modification (PTM) that change the fate of the underlying genomic information, right through to transcription hubs, and the recently elucidated roles of IDPs and IDRs in the condensation of large regions of the genome through liquid–liquid phase separation.

The dynamic structure of chromatin is linked to gene regulation and many other biological functions. Consequently, it is of importance to understand the factors that regulate chromatin dynamics. While the in vivo analysis of chromatin has verified that histone post-translational modifications play a role in modulating DNA accessibility, the complex nuclear environment and multiplicity of modifications prevents clear conclusions as to how individual modifications influence chromatin dynamics in the cell. For this reason, in vitro analyses of model reconstituted nucleosomal arrays has been pivotal in understanding the dynamic nature of chromatin compaction and the affects that specific post-translational modifications can have on the higher order chromatin structure. In this mini-review, we briefly describe the dynamic chromatin structures that have been observed in vitro and the environmental conditions that give rise to these various conformational states. Our focus then turns to a discussion of the specific histone post-translational modifications that have been shown to alter formation of these higher order chromatin structures in vitro and how this may relate to the biological state and accessibility of chromatin in vivo .

The DNA of eukaryotic chromatin and chromosomes is repeatedly supercoiled around histone octamers forming ‘beads-on-a-string’ chains of nucleosomes. The extent of nucleosome chain folding and DNA accessibility vary between different functional and epigenetic states of nuclear chromatin and change dramatically upon cell differentiation, but the molecular mechanisms that direct 3D folding of the nucleosome chain in vivo are still enigmatic. Recent advances in cell imaging and chromosome capture techniques have radically challenged the established paradigm of regular and hierarchical chromatin fibers by highlighting irregular chromatin organization and the importance of the nuclear skeletal structures hoisting the nucleosome chains. Here, we argue that, by analyzing individual structural elements of the nucleosome chain – nucleosome spacing, linker DNA conformations, internucleosomal interactions, and nucleosome chain flexibility – and integrating these elements in multiplex 3D structural models, we can predict the features of the multiplex chromatin folding assemblies underlying distinct developmental and epigenetic states in living cells. Furthermore, partial disassembly of the nuclear structures suspending chromatin fibers may reveal the intrinsic mechanisms of nucleosome chain folding. These mechanisms and structures are expected to provide molecular cues to modify chromatin structure and functions related to developmental and disease processes.

Due to genetic heterogeneity across patients, the identification of effective disease signatures and therapeutic targets is challenging. Addressing this challenge, we have previously developed a network-based approach, which integrates heterogeneous sources of biological information to identify disease specific core-regulatory networks. In particular, our workflow uses a multi-objective optimization function to calculate a ranking score for network components (e.g. feedback/feedforward loops) based on network properties, biomedical and high-throughput expression data. High ranked network components are merged to identify the core-regulatory network(s) that is then subjected to dynamical analysis using stimulus–response and in silico perturbation experiments for the identification of disease gene signatures and therapeutic targets. In a case study, we implemented our workflow to identify bladder and breast cancer specific core-regulatory networks underlying epithelial–mesenchymal transition from the E2F1 molecular interaction map. In this study, we review our workflow and described how it has developed over time to understand the mechanisms underlying disease progression and prediction of signatures for clinical decision making.

Gene regulatory networks control the cellular phenotype by changing the RNA and protein composition. Despite its importance, the gene regulatory network in higher organisms is only partly mapped out. Here, we investigate the potential of reverse engineering methods to unravel the structure of these networks. Particularly, we focus on modular response analysis (MRA), a method that can disentangle networks from perturbation data. We benchmark a version of MRA that was previously successfully applied to reconstruct a signalling-driven genetic network, termed MLMSMRA, to test cases mimicking various aspects of gene regulatory networks. We then investigate the performance in comparison with other MRA realisations and related methods. The benchmark shows that MRA has the potential to predict functional interactions, but also shows that successful application of MRA is restricted to small sparse networks and to data with a low signal-to-noise ratio.

The cellular abundance of proteins can vary even between isogenic single cells. This variability between single-cell protein levels can have regulatory roles, such as controlling cell fate during apoptosis induction or the proliferation/quiescence decision. Here, we review examples connecting protein levels and their dynamics in single cells to cellular functions. Such findings were made possible by the introduction of antibodies, and subsequently fluorescent proteins, for tracking protein levels in single cells. However, in heterogeneous cell populations, such as tumors or differentiating stem cells, cellular decisions are controlled by hundreds, even thousands of proteins acting in concert. Characterizing such complex systems demands measurements of thousands of proteins across thousands of single cells. This demand has inspired the development of new methods for single-cell protein analysis, and we discuss their trade-offs, with an emphasis on their specificity and coverage. We finish by highlighting the potential of emerging mass-spec methods to enable systems-level measurement of single-cell proteomes with unprecedented coverage and specificity. Combining such methods with methods for quantitating the transcriptomes and metabolomes of single cells will provide essential data for advancing quantitative systems biology.

Mitochondria are the major source of ATP in the cell. Five multi-subunit complexes in the inner membrane of the organelle are involved in the oxidative phosphorylation required for ATP production. Thirteen subunits of these complexes are encoded by the mitochondrial genome often referred to as mtDNA. For this reason, the expression of mtDNA is vital for the assembly and functioning of the oxidative phosphorylation complexes. Defects of the mechanisms regulating mtDNA gene expression have been associated with deficiencies in assembly of these complexes, resulting in mitochondrial diseases. Recently, numerous factors involved in these processes have been identified and characterized leading to a deeper understanding of the mechanisms that underlie mitochondrial diseases.

Mitochondrial DNA (mtDNA) is a multi-copy genome whose cell copy number varies depending on tissue type. Mutations in mtDNA can cause a wide spectrum of diseases. Mutated mtDNA is often found as a subset of the total mtDNA population in a cell or tissue, a situation known as heteroplasmy. As mitochondrial dysfunction only presents after a certain level of heteroplasmy has been acquired, ways to artificially reduce or replace the mutated species have been attempted. This review addresses recent approaches and advances in this field, focusing on the prevention of pathogenic mtDNA transfer via mitochondrial donation techniques such as maternal spindle transfer and pronuclear transfer in which mutated mtDNA in the oocyte or fertilized embryo is substituted with normal copies of the mitochondrial genome. This review also discusses the molecular targeting and cleavage of pathogenic mtDNA to shift heteroplasmy using antigenomic therapy and genome engineering techniques including Zinc-finger nucleases and transcription activator-like effector nucleases. Finally, it considers CRISPR technology and the unique difficulties that mitochondrial genome editing presents.

Extracellular vesicles (EVs) are produced by invading pathogens and also by host cells in response to infection. The origin, composition, and function of EVs made during infection are diverse and provide effective vehicles for localized and broad dissimilation of effector molecules in the infected host. Extracellular pathogens use EVs to communicate with each other by sensing the host environment contributing to social motility, tissue tropism, and persistence of infection. Pathogen-derived EVs can also interact with host cells to influence the adhesive properties of host membranes and to alter immune recognition and response. Intracellular pathogens can affect both the protein and RNA content of EVs produced by infected host cells. Release of pathogen-induced host EVs can affect host immune responses to infection. In this review, we will describe both the biogenesis and content of EVs produced by a number of diverse pathogens. In addition, we will examine the pathogen-induced changes to EVs produced by infected host cells.

Plastids are critical organelles in plant cells that perform diverse functions and are central to many metabolic pathways. Beyond their major roles in primary metabolism, of which their role in photosynthesis is perhaps best known, plastids contribute to the biosynthesis of phytohormones and other secondary metabolites, store critical biomolecules, and sense a range of environmental stresses. Accordingly, plastid-derived signals coordinate a host of physiological and developmental processes, often by emitting signalling molecules that regulate the expression of nuclear genes. Several excellent recent reviews have provided broad perspectives on plastid signalling pathways. In this review, we will highlight recent advances in our understanding of chloroplast signalling pathways. Our discussion focuses on new discoveries illuminating how chloroplasts determine life and death decisions in cells and on studies elucidating tetrapyrrole biosynthesis signal transduction networks. We will also examine the role of a plastid RNA helicase, ISE2, in chloroplast signalling, and scrutinize intriguing results investigating the potential role of stromules in conducting signals from the chloroplast to other cellular locations.

In contrast to the cyanobacterial ancestor, chloroplast gene expression is predominantly governed on the post-transcriptional level such as modifications of the RNA sequence, decay rates, exo- and endonucleolytic processing as well as translational events. The concerted function of numerous chloroplast RNA-binding proteins plays a fundamental and often essential role in all these processes but our understanding of their impact in regulation of RNA degradation is only at the beginning. Moreover, metabolic processes and post-translational modifications are thought to affect the function of RNA protectors. These protectors contain a variety of different RNA-recognition motifs, which often appear as multiple repeats. They are required for normal plant growth and development as well as diverse stress responses and acclimation processes. Interestingly, most of the protectors are plant specific which reflects a fast-evolving RNA metabolism in chloroplasts congruent with the diverging RNA targets. Here, we mainly focused on the characteristics of known chloroplast RNA-binding proteins that protect exonuclease-sensitive sites in chloroplasts of vascular plants.

Although chloroplasts contain their own genetic system and are semi-autonomous cell organelles, plastid biogenesis and homeostasis are heavily dependent on the nucleo-cytosolic compartment. These two cellular compartments are closely co-ordinated through a complex signaling network comprising both anterograde and retrograde signaling chains. Developmental changes or any perturbation in the chloroplast system induced by a particular stress resulting from changes in environmental conditions such as excess light, elevated temperature, nutrient limitation, pathogen infection, give rise to specific signals. They migrate out of the chloroplast and are perceived by the nucleus where they elicit changes in expression of particular genes that allow for the maintenance of plastid homeostasis toward environmental cues. These genes mainly include those of photosynthesis-associated proteins, chaperones, proteases, nucleases and immune/defense proteins. Besides this transcriptional response, a chloroplast quality control system exists that is involved in the repair and turnover of damaged plastid proteins. This system degrades aggregated or damaged proteins and it can even remove entire chloroplasts when they have suffered heavy damage. This response comprises several processes such as plastid autophagy and ubiquitin–proteasome mediated proteolysis that occurs on the plastid envelope through the action of the ubiquitin–proteasome system.

Ageing can be defined as the gradual deterioration of physiological functions, increasing the incidence of age-related disorders and the probability of death. Therefore, the term ageing not only reflects the lifespan of an organism but also refers to progressive functional impairment and disease. The nutrient-sensing kinase mTOR (mammalian target of rapamycin) is a major determinant of ageing. mTOR promotes cell growth and controls central metabolic pathways including protein biosynthesis, autophagy and glucose and lipid homoeostasis. The concept that mTOR has a crucial role in ageing is supported by numerous reports on the lifespan-prolonging effects of the mTOR inhibitor rapamycin in invertebrate and vertebrate model organisms. Dietary restriction increases lifespan and delays ageing phenotypes as well and mTOR has been assigned a major role in this process. This may suggest a causal relationship between the lifespan of an organism and its metabolic phenotype. More than 25 years after mTOR’s discovery, a wealth of metabolic and ageing-related effects have been reported. In this review, we cover the current view on the contribution of the different elements of the mTOR signalling network to lifespan and age-related metabolic impairment. We specifically focus on distinct roles of isoforms and splice variants across the mTOR network. The comprehensive analysis of mouse knockout studies targeting these variants does not support a tight correlation between lifespan prolongation and improved metabolic phenotypes and questions the strict causal relationship between them.

DNA is the carrier of genetic information and the primary template from which all cellular information is ultimately derived. Changes in the DNA information content through mutation generate diversity for evolution through natural selection but are also a source of deleterious effects. It has since long been hypothesized that mutation accumulation in somatic cells of multicellular organisms could causally contribute to age-related cellular degeneration and death. Assays to detect different types of mutations, from base substitutions to large chromosomal aberrations, have been developed and show unequivocally that mutations accumulate in different tissues and cell types of ageing humans and animals. More recently, next-generation sequencing-based methods have been developed to accurately determine the complete landscape of base substitution mutations in single cells. The first results show that the somatic mutation rate is much higher than the germline mutation rate and that base substitution loads in somatic cells are high enough to potentially affect cellular function.

Acquisition of antibiotic resistance is a relevant problem for human health. The selection and spread of antibiotic-resistant organisms not only compromise the treatment of infectious diseases, but also the implementation of different therapeutic procedures as organ transplantation, advanced surgery or chemotherapy, all of which require proficient methods for avoiding infections. It has been generally accepted that the acquisition of antibiotic resistance will produce a general metabolic burden: in the absence of selection, the resistant organisms would be outcompeted by the susceptible ones. If that was always true, discontinuation of antibiotic use would render the disappearance of resistant microorganisms. However, several studies have shown that, once resistance emerges, the recovery of a fully susceptible population even in the absence of antibiotics is not easy. In the present study, we review updated information on the effect of the acquisition of antibiotic resistance in bacterial physiology as well as on the mechanisms that allow the compensation of the fitness costs associated with the acquisition of resistance.

Cells have developed robust adaptation mechanisms to survive environmental conditions that challenge the integrity of their proteome and ensure cellular viability. These are stress signalling pathways that integrate extracellular signals with the ability to detect and efficiently respond to protein-folding perturbations within the cell. Within the context of an organism, the cell-autonomous effects of these signalling mechanisms are superimposed by cell-non-autonomous stress signalling pathways that allow co-ordination of stress responses across tissues. These transcellular stress signalling pathways orchestrate and maintain the cellular proteome at an organismal level. This article focuses on mechanisms in both invertebrate and vertebrate organisms that activate stress responses in a cell-non-autonomous manner. We discuss emerging insights and provide specific examples on how components of the cell-non-autonomous proteostasis network are used in cancer and protein-folding diseases to drive disease progression across tissues.