Mammalian Nucleus Research Articles

Biology and Physics of Heterochromatin-Like Domains/Complexes.

The hallmarks of constitutive heterochromatin, HP1 and H3K9me2/3, assemble heterochromatin-like domains/complexes outside canonical constitutively heterochromatic territories where they regulate chromatin template-dependent processes. Domains are more than 100 kb in size; complexes less than 100 kb. They are present in the genomes of organisms ranging from fission yeast to human, with an expansion in size and number in mammals. Some of the likely functions of domains/complexes include silencing of the donor mating type region in fission yeast, preservation of DNA methylation at imprinted germline differentially methylated regions (gDMRs) and regulation of the phylotypic progression during vertebrate development. Far cis- and trans-contacts between micro-phase separated domains/complexes in mammalian nuclei contribute to the emergence of epigenetic compartmental domains (ECDs) detected in Hi-C maps. A thermodynamic description of micro-phase separation of heterochromatin-like domains/complexes may require a gestalt shift away from the monomer as the “unit of incompatibility” that determines the sign and magnitude of the Flory–Huggins parameter, χ. Instead, a more dynamic structure, the oligo-nucleosomal “clutch”, consisting of between 2 and 10 nucleosomes is both the long sought-after secondary structure of chromatin and its unit of incompatibility. Based on this assumption we present a simple theoretical framework that enables an estimation of χ for domains/complexes flanked by euchromatin and thereby an indication of their tendency to phase separate. The degree of phase separation is specified by χN, where N is the number of “clutches” in a domain/complex. Our approach could provide an additional tool for understanding the biophysics of the 3D genome.

3150 – EPIGENETIC REGULATOR ASXL1 PROMOTES PARASPECKLE FORMATION THROUGH C-TERMINAL INTRINSICALLY DISORDERED REGION IN HEMATOPOIETIC CELLS

The importance of membraneless organelles and these formation mechanisms, liquid-liquid phase separation (LLPS) has recently gained large attention due to new understandings on their roles in cellular functions including transcription, RNA splicing and signal transduction. Paraspeckles are well-known membraneless organelless located in the interchromatin space of mammalian cell nuclei and consist of multiple proteins and RNAs, including NONO, SFPQ and NEAT1. These paraspeckle components are highly expressed in hematopoietic cells. However, their roles in hematopoiesis are largely unknown. In this study, we found an unexpected involvement of epigenetic regulator ASXL1 in paraspeckle formation in hematopoietic cells. ASXL1 has a long intrinsically disordered region (IDR) in its C-terminal region and forms phase-separated droplets in vitro. Of note, a large part of the IDR is located behind the mutational hotspot of ASXL1 gene, suggesting that most pathogenic ASXL1mutants lack IDR. Interestingly, the mutant ASXL1 (ASXL1-MT) lacking its C-terminal IDR could not form droplets efficiently. ASXL1 upregulates NEAT1 expression and increases NONO-NEAT1 interaction, while a pathogenic ASXL1-MT does not support the interaction among paraspeckle components. Consequently, we observed disruption of paraspeckles and aberrant localization of Nono in cytoplasm of hematopoietic stem and progenitor cells (HSPCs) derived from ASXL1-MT knockin mice. We also showed that NONO depletion and forced expression of cytoplasmic NONO impair repopulating potential of HSPCs as ASXL1-MT does. The present findings highlight the potentially important role of paraspeckles in hematopoiesis and hematopoietic disorders.

Neuronal and Non-neuronal Cell Types Displaying Circadian Rhythmicity in the Mammalian Suprachiasmatic Nucleus.

Neuronal and Non-neuronal Cell Types Displaying Circadian Rhythmicity in the Mammalian Suprachiasmatic Nucleus.

Postmitotic Hoxa5 Expression Specifies Pontine Neuron Positional Identity and Input Connectivity of Cortical Afferent Subsets

The mammalian precerebellar pontine nucleus (PN) has a main role in relaying cortical information to the cerebellum. The molecular determinants establishing ordered connectivity patterns between cortical afferents and precerebellar neurons are largely unknown. We show that expression of Hox5 transcription factors is induced in specific subsets of postmitotic PN neurons at migration onset. Hox5 induction is achieved by response to retinoic acid signaling, resulting in Jmjd3-dependent derepression of Polycomb chromatin and 3D conformational changes. Hoxa5 drives neurons to settle posteriorly in the PN, where they are monosynaptically targeted by cortical neuron subsets mainly carrying limb somatosensation. Furthermore, Hoxa5 postmigratory ectopic expression in PN neurons is sufficient to attract cortical somatosensory inputs regardless of position and avoid visual afferents. Transcriptome analysis further suggests that Hoxa5 is involved in circuit formation. Thus, Hoxa5 coordinates postmitotic specification, migration, settling position, and sub-circuit assembly of PN neuron subsets in the cortico-cerebellar pathway.

Transcription hub dynamics revealed by nascent RNA‐chromatin interactomes

In mammalian nucleus, many types of RNA are physically associated with chromatin. Specific and diverse functions have been increasingly ascribed to these chromatin‐associated RNAs, particularly in transcription and organization of 3‐dimensional chromatin architecture. Yet the full scope of molecular mechanisms that chromatin‐associated RNAs employ is far from understood. These mechanisms may be key to delineating regulatory redundancy and plasticity of transcription factors and enhancers. There is thus a compelling need to integrate chromatin‐associated RNA into our understanding of gene expression regulation. Among these chromatin‐associated RNA species, over 85% are nascent mRNAs being tethered to their sites of transcription. These nascent transcripts form individual ‘clouds’ over regulatory elements in cis. Such clouds enable faithful depiction of the landscape of transcription hubs. While decoration by these clouds of nascent mRNA on chromatin seems passive, evidence has indicated that it may dictate the expression of neighboring genes within the hubs. We therefore hypothesize that, before traveling to the cytoplasm as the messengers for protein synthesis, nascent mRNAs may modulate gene expression in the nucleus via interacting with chromatin. Two distinct types of interplay exist between nascent mRNA and the local transcription activity have been identified. In the RNA‐dependent model, nascent transcripts reinforce the local transcription microenvironment to promote expression of itself and neighboring gene(s). In the transcription‐dependent model, neighboring genes within a hub compete for enhancer activation and thereby repress the expression of one another. These findings underscore the importance of nascent mRNAs in their regulatory roles outside of the central dogma, adding a new layer to the paradigm in gene expression regulation.

Single-molecule displacement mapping unveils nanoscale heterogeneities in intracellular diffusivity.

Intracellular diffusion underlies vital cellular processes. However, it remains difficult to elucidate how an unbound protein diffuses inside the cell with good spatial resolution and sensitivity. Here we introduce single-molecule displacement/diffusivity mapping (SMdM), a super-resolution strategy that enables the nanoscale mapping of intracellular diffusivity through local statistics of the instantaneous displacements of freely diffusing single molecules. We thus show that the diffusion of an average-sized protein in the mammalian cytoplasm and nucleus to both be spatially heterogeneous at the nanoscale, and such variations in local diffusivity correlate with the ultrastructure of the actin cytoskeleton and the chromosome, respectively. SMdM of differently charged proteins further unveils that the possession of positive, but not negative, net charges drastically impedes diffusion, and that the exact degree of slowdown is determined by the specific subcellular environments. We thus open a new door to probing intracellular properties and functions at the nanoscale.

Integration of Swimming-Related Synaptic Excitation and Inhibition by olig2+ Eurydendroid Neurons in Larval Zebrafish Cerebellum.

The cerebellum influences motor control through Purkinje target neurons, which transmit cerebellar output. Such output is required, for instance, for larval zebrafish to learn conditioned fictive swimming. The output cells, called eurydendroid neurons (ENs) in teleost fish, are inhibited by Purkinje cells and excited by parallel fibers. Here, we investigated the electrophysiological properties of glutamatergic ENs labeled by the transcription factor olig2. Action potential firing and synaptic responses were recorded in current clamp and voltage clamp from olig2+ neurons in immobilized larval zebrafish (before sexual differentiation) and were correlated with motor behavior by simultaneous recording of fictive swimming. In the absence of swimming, olig2+ ENs had basal firing rates near 8 spikes/s, and EPSCs and IPSCs were evident. Comparing Purkinje firing rates and eurydendroid IPSC rates indicated that 1-3 Purkinje cells converge onto each EN. Optogenetically suppressing Purkinje simple spikes, while preserving complex spikes, suggested that eurydendroid IPSC size depended on presynaptic spike duration rather than amplitude. During swimming, EPSC and IPSC rates increased. Total excitatory and inhibitory currents during sensory-evoked swimming were both more than double those during spontaneous swimming. During both spontaneous and sensory-evoked swimming, the total inhibitory current was more than threefold larger than the excitatory current. Firing rates of ENs nevertheless increased, suggesting that the relative timing of IPSCs and EPSCs may permit excitation to drive additional eurydendroid spikes. The data indicate that olig2+ cells are ENs whose activity is modulated with locomotion, suiting them to participate in sensorimotor integration associated with cerebellum-dependent learning.SIGNIFICANCE STATEMENT The cerebellum contributes to movements through signals generated by cerebellar output neurons, called eurydendroid neurons (ENs) in fish (cerebellar nuclei in mammals). ENs receive sensory and motor signals from excitatory parallel fibers and inhibitory Purkinje cells. Here, we report electrophysiological recordings from ENs of larval zebrafish that directly illustrate how synaptic inhibition and excitation are integrated by cerebellar output neurons in association with motor behavior. The results demonstrate that inhibitory and excitatory drive both increase during fictive swimming, but inhibition greatly exceeds excitation. Firing rates nevertheless increase, providing evidence that synaptic integration promotes cerebellar output during locomotion. The data offer a basis for comparing aspects of cerebellar coding that are conserved and that diverge across vertebrates.

Radial Organization in the Mammalian Nucleus.

In eukaryotic cells, most of the genetic material is contained within a highly specialized organelle—the nucleus. A large body of evidence indicates that, within the nucleus, chromatinized DNA is spatially organized at multiple length scales. The higher-order organization of chromatin is crucial for proper execution of multiple genome functions, including DNA replication and transcription. Here, we review our current knowledge on the spatial organization of chromatin in the nucleus of mammalian cells, focusing in particular on how chromatin is radially arranged with respect to the nuclear lamina. We then discuss the possible mechanisms by which the radial organization of chromatin in the cell nucleus is established. Lastly, we propose a unifying model of nuclear spatial organization, and suggest novel approaches to test it.

Ovarian hormones induce de novo DNA methyltransferase expression in the Siberian hamster suprachiasmatic nucleus.

The present study investigated neuroanatomically localised changes in de novo DNA methyltransferase expression in the female Siberian hamster (Phodopus sungorus). The objectives were to identify the neuroendocrine substrates that exhibit rhythmic Dnmt3a and Dnmt3b expression across the oestrous cycle and also examine the role of ovarian steroids. Hypothalamic Dnmt3a expression was observed to significantly increase during the transition from pro-oestrous to oestrous. A single bolus injection of diethylstilbestrol and progesterone was sufficient to increase Dnmt3a cell numbers and Dnmt3b immunoreactive intensity in the suprachiasmatic nucleus. In vitro analyses using an embryonic rodent cell line revealed that diethylstilbestrol was sufficient to induce Dnmt3b expression. Up-regulating DNA methylation in vitro reduced the expression of vasoactive intestinal polypeptide, Vip, and the circadian clock gene, Bmal1. Together, these data indicate that ovarian steroids drive de novo DNA methyltransferase expression in the mammalian suprachiasmatic nucleus and increased methylation may regulate genes involved in the circadian timing of oestrous: Vip and Bmal1. Overall, epigenetically mediated neuroendocrine reproductive events may reflect an evolutionarily ancient process involved in the timing of female fertility.

Biophysical Characterization of G-Quadruplex Structure in Long Noncoding RNA NEAT1

Paraspeckles are structures found in the interchromatin space of mammalian nuclei and are a relatively newly identified subnuclear body whose function is still largely unknown. Originally identified as being comprised of different paraspeckle proteins, it was later discovered that paraspeckles also contained RNA. Nuclear enriched abundant transcript 1 (NEAT1) is a long noncoding RNA (lncRNA) that has two different isoforms – NEAT1_1 and NEAT1_2, both of which are components of the paraspeckle. However, only NEAT1_2, consisting of around 22,700 nucleotides, has been found to be an essential component of paraspeckles, with each paraspeckle containing up to 50 NEAT1_2 molecules. FUS, a protein associated with amyotrophic lateral sclerosis (ALS), has been shown to be indispensable in the formation of paraspeckles, as these structures are not observed in FUS KO cells. FUS has been proposed to be implicated in maintaining the paraspeckle structure through its interactions with NEAT1; however, the molecular principles of recognition between FUS and NEAT1 are not known. FUS has been shown to bind G-quadruplex structures and in this study, we used biophysical methods such as NMR, CD, and UV spectroscopy to investigate if NEAT1 can form G-quadruplex structures that could be potentially recognized by FUS.

Oxidation of Sperm Nucleus in Mammals: A Physiological Necessity to Some Extent with Adverse Impacts on Oocyte and Offspring.

Sperm cells have long been known to be good producers of reactive oxygen species, while they are also known to be particularly sensitive to oxidative damage affecting their structures and functions. As with all organic cellular components, sperm nuclear components and, in particular, nucleic acids undergo oxidative alterations that have recently been shown to be commonly encountered in clinical practice. This review will attempt to provide an overview of this situation. After a brief coverage of the biological reasons why the sperm nucleus and associated DNA are sensitive to oxidative damage, a summary of the most recent results concerning the oxidation of sperm DNA in animal and human models will be presented. The study will then attempt to cover the possible consequences of sperm nuclear oxidation on male fertility and beyond.

Recent advances in the spatial organization of the mammalian genome

The mammalian genome is complex and presents a dynamic structural organization that reflects function. Organization of the genome inside the mammalian nucleus impacts all nuclear processes including but not limited to transcription, replication and repair, and in many biological contexts such as early development, differentiation and physiological adaptations. However, there is limited understating of how 3D organization of the mammalian genome regulates different nuclear processes. Recent advances in microscopy and a myriad of genomics methods -- ropelled by next-generation sequencing -- have advanced our knowledge of genome organization to a great extent. In this review, we discuss nuclear compartments in general and recent advances in the understanding of how mammalian genome is organized in these compartments with an emphasis on dynamics at the nuclear periphery.

Characterization of Plant 3D Chromatin Architecture, In Situ Hi-C Library Preparation, and Data Analysis.

In the mammalian nucleus, 3D organization of the chromatin plays an important role in the regulation of gene expression. Similar chromatin structures such as A/B compartments, domains, and loops have now been found in plants, yet their biological function remained to be further elucidated. In this chapter, we present a detailed protocol for examining genome-wide chromatin interaction using in situ Hi-C optimized for plant tissues. We also provide a step-by-step bioinformatic workflow for Hi-C sequencing data alignment, and subsequent compartment, domain, and chromatin loop identification.

Refinement of the dopaminergic system of anuran amphibians based on connectivity with habenula, basal ganglia, limbic system, pallium, and spinal cord.

Whereas our understanding of the dopaminergic system in mammals allows for a distinction between ventral tegmental area (VTA) and substantia nigra pars compacta (SNc), no clear evidence for separate structures in anamniotes has been presented to date. To broaden the insight into the organization and regulation of neuromodulatory systems in anuran amphibians, tracing and immunohistochemical investigations were performed in the Oriental fire-bellied toad, Bombina orientalis. Topographically organized catecholaminergic "nigrostriatal," "mesolimbic," "mesocortical," and spinal cord projections arising from the posterior tubercle and mesencephalic tegmentum were identified. We compared these results with published data from lampreys, chondrichthyes, teleosts, amphibians, reptiles, birds, and mammals. Based on the pattern of organization, as well as the differential innervation by the habenular nuclei, domains gradually comparable to the mammalian paranigral VTA, ventral tier of the SNc, interfascicular nucleus of the VTA, and supramamillary/retromamillary area were identified. Additionally, we could demonstrate topographic separate populations of habenula neurons projecting via a direct excitatory or indirect GABAergic pathway onto the catecholaminergic VTA/SNc homologs and serotonergic raphe nuclei. The indirect GABAergic habenula pathway derives from neurons in the superficial mamillary area, which in terms of its connectivity and chemoarchitecture resembles the mammalian rostromedial tegmental nucleus. These results demonstrate a much more elaborate interconnection principle of the anuran dopaminergic system than previously assumed. Based on the data presented it seems that most features of the dopaminergic system of amniotes had already evolved when the amphibian line of evolution diverged from that leading up to mammals, reptiles, and birds.

Polymer Modeling Predicts Chromosome Reorganization in Senescence

SummaryLamina-associated domains (LADs) cover a large part of the human genome and are thought to play a major role in shaping the nuclear architectural landscape. Here, we perform polymer simulations, microscopy, and mass spectrometry to dissect the roles played by heterochromatin- and lamina-mediated interactions in nuclear organization. Our model explains the conventional organization of heterochromatin and euchromatin in growing cells and the pathological organization found in oncogene-induced senescence and progeria. We show that the experimentally observed changes in the locality of contacts in senescent and progeroid cells can be explained as arising due to phase transitions in the system. Within our simulations, LADs are highly stochastic, as in experiments. Our model suggests that, once established, the senescent phenotype should be metastable even if lamina-mediated interactions were reinstated. Overall, our simulations uncover a generic physical mechanism that can regulate heterochromatin segregation and LAD formation in a wide range of mammalian nuclei.

Pulvinar Modulates Contrast Responses in the Visual Cortex as a Function of Cortical Hierarchy.

The pulvinar is the largest extrageniculate visual nucleus in mammals. Given its extensive reciprocal connectivity with the visual cortex, it allows the cortico-thalamocortical transfer of visual information. Nonetheless, knowledge of the nature of the pulvinar inputs to the cortex remains elusive. We investigated the impact of silencing the pulvinar on the contrast response function of neurons in 2 distinct hierarchical cortical areas in the cat (areas 17 and 21a). Pulvinar inactivation altered the response gain in both areas, but with larger changes observed in area 21a. A theoretical model was proposed, simulating the pulvinar contribution to cortical contrast responses by modifying the excitation-inhibition balanced state of neurons across the cortical hierarchy. Our experimental and theoretical data showed that the pulvinar exerts a greater modulatory influence on neuronal activity in area 21a than in the primary visual cortex, indicating that the pulvinar impact on cortical visual neurons varies along the cortical hierarchy.

Statistics of chromatin organization during cell differentiation revealed by heterogeneous cross-linked polymers

Chromatin of mammalian nucleus folds into discrete contact enriched regions such as Topologically Associating Domains (TADs). Folding hierarchy and internal organization of TADs is highly dynamic throughout cellular differentiation, and are correlated with gene activation and silencing. To account for multiple interacting TADs, we developed a parsimonious randomly cross-linked (RCL) polymer model that maps high frequency Hi-C encounters within and between TADs into direct loci interactions using cross-links at a given base-pair resolution. We reconstruct three TADs of the mammalian X chromosome for three stages of differentiation. We compute the radius of gyration of TADs and the encounter probability between genomic segments. We found 1) a synchronous compaction and decompaction of TADs throughout differentiation and 2) high order organization into meta-TADs resulting from weak inter-TAD interactions. Finally, the present framework allows to infer transient properties of the chromatin from steady-state statistics embedded in the Hi-C/5C data.

Varying levels of X chromosome coalescence in female somatic cells alters the balance of X-linked dosage compensation and is implicated in female-dominant systemic lupus erythematosus

The three-dimensional organization of the genome in mammalian interphase nuclei is intrinsically linked to the regulation of gene expression. Whole chromosome territories and their encoded gene loci occupy preferential positions within the nucleus that changes according to the expression profile of a given cell lineage or stage. To further illuminate the relationship between chromosome organization, epigenetic environment, and gene expression, here we examine the functional organization of chromosome X and corresponding X-linked genes in a variety of healthy human and disease state X diploid (XX) cells. We observe high frequencies of homologous chromosome X colocalization (or coalescence), typically associated with initiation of X-chromosome inactivation, occurring in XX cells outside of early embryogenesis. Moreover, during chromosome X coalescence significant changes in Xist, H3K27me3, and X-linked gene expression occur, suggesting the potential exchange of gene regulatory information between the active and inactive X chromosomes. We also observe significant differences in chromosome X coalescence in disease-implicated lymphocytes isolated from systemic lupus erythematosus (SLE) patients compared to healthy controls. These results demonstrate that X chromosomes can functionally interact outside of embryogenesis when X inactivation is initiated and suggest a potential gene regulatory mechanism aberration underlying the increased frequency of autoimmunity in XX individuals.

Molecular anatomy of the architectural NEAT1 noncoding RNA: The domains, interactors, and biogenesis pathway required to build phase-separated nuclear paraspeckles.

Long noncoding RNAs (lncRNAs) are extremely diverse and have various significant physiological functions. lncRNAs generally associate with specific sets of RNA-binding proteins (RBPs) to form functional ribonucleoprotein (RNP) complexes. NEAT1 is a highly abundant lncRNA in the mammalian cell nucleus that associates with specific RBPs to form NEAT1 RNPs. Intriguingly, cellular NEAT1 RNPs are extraordinarily large and can be detected using an optical microscope. These gigantic RNPs, so-called paraspeckles, are a type of membraneless nuclear body. Paraspeckles contain approximately 50 NEAT1 RNA molecules together with characteristic RBPs possessing aggregation-prone prion-like domains. Paraspeckle formation proceeds on the nascent NEAT1 transcript in conjunction with NEAT1 biogenesis, which exhibits various features that differ from those exhibited by mRNA biogenesis, including a lack of introns, noncanonical 3' end formation, and nuclear retention. These unique features may be required for the mechanism of paraspeckle formation. NEAT1 possesses three distinct RNA domains (A, B, and C), which function in stabilization (A), isoform switching (B), and paraspeckle assembly (C). In particular, the central C domain contains smaller subdomains that are high-affinity binding sites for the essential paraspeckle proteins (NONO and SFPQ) that subsequently polymerize along NEAT1. Subsequent recruitment of additional essential PSPs (FUS and RBM14) induces liquid-liquid phase separation to build a massive paraspeckle structure. Thus, the molecular anatomy of the NEAT1 arcRNA provides an ideal model to understand how lncRNAs form the functional RNP machinery. This article is characterized under: RNA Export and Localization > Nuclear Export/Import RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Phase-to-Phase With Nucleoli - Stress Responses, Protein Aggregation and Novel Roles of RNA.

Protein- and RNA-containing foci and aggregates are a hallmark of many age- and mutation-related neurodegenerative diseases. This article focuses on the role the nucleolus has as a hub in macromolecule regulation in the mammalian nucleus. The nucleolus has a well-established role in ribosome biogenesis and functions in several types of cellular stress responses. In addition to known reactions to DNA damaging and transcription inhibiting stresses, there is an emerging role of the nucleolus especially in responses to proteotoxic stress such as heat shock and inhibition of proteasome function. The nucleolus serves as an active regulatory site for detention of extranucleolar proteins. This takes place in nucleolar cavities and manifests in protein and RNA collections referred to as intranucleolar bodies (INBs), nucleolar aggresomes or amyloid bodies (A-bodies), depending on stress type, severity of accumulation, and material propensities of the macromolecular collections. These indicate a relevance of nucleolar function and regulation in neurodegeneration-related cellular events, but also provide surprising connections with cancer-related pathways. Yet, the molecular mechanisms governing these processes remain largely undefined. In this article, the nucleolus as the site of protein and RNA accumulation and as a possible protective organelle for nuclear proteins during stress is viewed. In addition, recent evidence of liquid-liquid phase separation (LLPS) and liquid-solid phase transition in the formation of nucleoli and its stress responses, respectively, are discussed, along with the increasingly indicated role and open questions for noncoding RNA species in these events.