Something silent this way forms: the functional organization of the repressive nuclear compartment.

The repressive compartment of the nucleus is comprised primarily of telomeric and centromeric regions, the silent portion of ribosomal RNA genes, the majority of transposable element repeats, and facultatively repressed genes specific to different cell types. This compartment localizes into three main regions: the peripheral heterochromatin, perinucleolar heterochromatin, and pericentromeric heterochromatin. Both chromatin remodeling proteins and transcription of noncoding RNAs are involved in maintenance of repression in these compartments. Global reorganization of the repressive compartment occurs at each cell division, during early development, and during terminal differentiation. Differential action of chromatin remodeling complexes and boundary element looping activities are involved in mediating these organizational changes. We discuss the evidence that heterochromatin formation and compartmentalization may drive nuclear organization.

Corrigendum: The 4D nucleome project.

This corrects the article DOI: 10.1038/nature23884.

The 4D nucleome project.

The 4D Nucleome Network aims to develop and apply approaches to map the structure and dynamics of the human and mouse genomes in space and time with the goal of gaining deeper mechanistic insights into how the nucleus is organized and functions. The project will develop and benchmark experimental and computational approaches for measuring genome conformation and nuclear organization, and investigate how these contribute to gene regulation and other genome functions. Validated experimental technologies will be combined with biophysical approaches to generate quantitative models of spatial genome organization in different biological states, both in cell populations and in single cells.

MicroRNAs with a nucleolar location.

There is increasing evidence that noncoding RNAs play a functional role in the nucleus. We previously reported that the microRNA (miRNA), miR-206, is concentrated in the nucleolus of rat myoblasts, as well as in the cytoplasm as expected. Here we have extended this finding. We show by cell/nuclear fractionation followed by microarray analysis that a number of miRNAs can be detected within the nucleolus of rat myoblasts, some of which are significantly concentrated there. Pronounced nucleolar localization is a specific phenomenon since other miRNAs are present at only very low levels in the nucleolus and occur at much higher levels in the nucleoplasm and/or the cytoplasm. We have further characterized a subset of these miRNAs using RT-qPCR and in situ hybridization, and the results suggest that some miRNAs are present in the nucleolus in precursor form while others are present as mature species. Furthermore, we have found that these miRNAs are clustered in specific sites within the nucleolus that correspond to the classical granular component. One of these miRNAs is completely homologous to a portion of a snoRNA, suggesting that it may be processed from it. In contrast, the other nucleolar-concentrated miRNAs do not show homology with any annotated rat snoRNAs and thus appear to be present in the nucleolus for other reasons, such as modification/processing, or to play roles in the late stages of ribosome biosynthesis or in nonribosomal functions that have recently been ascribed to the granular component of the nucleolus.

HP1α is a chromatin crosslinker that controls nuclear and mitotic chromosome mechanics.

Chromatin, which consists of DNA and associated proteins, contains genetic information and is a mechanical component of the nucleus. Heterochromatic histone methylation controls nucleus and chromosome stiffness, but the contribution of heterochromatin protein HP1α (CBX5) is unknown. We used a novel HP1α auxin-inducible degron human cell line to rapidly degrade HP1α. Degradation did not alter transcription, local chromatin compaction, or histone methylation, but did decrease chromatin stiffness. Single-nucleus micromanipulation reveals that HP1α is essential to chromatin-based mechanics and maintains nuclear morphology, separate from histone methylation. Further experiments with dimerization-deficient HP1αI165E indicate that chromatin crosslinking via HP1α dimerization is critical, while polymer simulations demonstrate the importance of chromatin-chromatin crosslinkers in mechanics. In mitotic chromosomes, HP1α similarly bolsters stiffness while aiding in mitotic alignment and faithful segregation. HP1α is therefore a critical chromatin-crosslinking protein that provides mechanical strength to chromosomes and the nucleus throughout the cell cycle and supports cellular functions.

Signal recognition particle RNA localization within the nucleolus differs from the classical sites of ribosome synthesis.

The nucleolus is the site of ribosome biosynthesis, but is now known to have other functions as well. In the present study we have investigated how the distribution of signal recognition particle (SRP) RNA within the nucleolus relates to the known sites of ribosomal RNA synthesis, processing, and nascent ribosome assembly (i.e., the fibrillar centers, the dense fibrillar component (DFC), and the granular component). Very little SRP RNA was detected in fibrillar centers or the DFC of the nucleolus, as defined by the RNA polymerase I-specific upstream binding factor and the protein fibrillarin, respectively. Some SRP RNA was present in the granular component, as marked by the protein B23, indicating a possible interaction with ribosomal subunits at a later stage of maturation. However, a substantial portion of SRP RNA was also detected in regions of the nucleolus where neither B23, UBF, or fibrillarin were concentrated. Dual probe in situ hybridization experiments confirmed that a significant fraction of nucleolar SRP RNA was not spatially coincident with 28S ribosomal RNA. These results demonstrate that SRP RNA concentrates in an intranucleolar location other than the classical stations of ribosome biosynthesis, suggesting that there may be nucleolar regions that are specialized for other functions.

Rapid, diffusional shuttling of poly(A) RNA between nuclear speckles and the nucleoplasm.

Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which caged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckle or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless of whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactivation-tracking studies, a temperature reduction from 37 to 22 degrees C had no discernible effect on the behavior of poly(A) RNA in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require metabolic energy.

A nonribosomal landscape in the nucleolus revealed by the stem cell protein nucleostemin.

Nucleostemin is a p53-interactive cell cycle progression factor that shuttles between the nucleolus and nucleoplasm, but it has no known involvement in ribosome synthesis. We found the dynamic properties of nucleostemin differed strikingly from fibrillarin (a protein directly involved in rRNA processing) both in response to rRNA transcription inhibition and in the schedule of reentry into daughter nuclei and the nucleolus during late telophase/early G1. Furthermore, nucleostemin was excluded from the nucleolar domains in which ribosomes are born--the fibrillar centers and dense fibrillar component. Instead it was concentrated in rRNA-deficient sites within the nucleolar granular component. This finding suggests that the nucleolus may be more subcompartmentalized than previously thought. In support of this concept, electron spectroscopic imaging studies of the nitrogen and phosphorus distribution in the nucleolar granular component revealed regions that are very rich in protein and yet devoid of nucleic acid. Together, these results suggest that the ultrastructural texture of the nucleolar granular component represents not only ribosomal particles but also RNA-free zones populated by proteins or protein complexes that likely serve other functions.

Diffusion-based transport of nascent ribosomes in the nucleus.

Although the complex process of ribosome assembly in the nucleolus is beginning to be understood, little is known about how the ribosomal subunits move from the nucleolus to the nuclear membrane for transport to the cytoplasm. We show here that large ribosomal subunits move out from the nucleolus and into the nucleoplasm in all directions, with no evidence of concentrated movement along directed paths. Mobility was slowed compared with that expected in aqueous solution in a manner consistent with anomalous diffusion. Once nucleoplasmic, the subunits moved in the same random manner and also sometimes visited another nucleolus before leaving the nucleus.

RNA polymerase III transcripts and the PTB protein are essential for the integrity of the perinucleolar compartment.

The perinucleolar compartment (PNC) is a nuclear substructure present in transformed cells. The PNC is defined by high concentrations of certain RNA binding proteins and a subset of small RNAs transcribed by RNA polymerase III (pol III), including the signal recognition particle RNA and an Alu RNA as reported here. To determine if the PNC is dependent on pol III transcription, HeLa cells were microinjected with the selective pol III inhibitor, Tagetin. This resulted in disassembly of the PNC, whereas inhibition of pol I by cycloheximide or pol II by alpha-amanitin did not significantly affect the PNC. However, overexpression of one of the PNC-associated RNAs from a pol II promoter followed by injection of Tagetin blocked the Tagetin-induced PNC disassembly, demonstrating that it is the RNA rather than pol III activity that is important for the PNC integrity. To elucidate the role of the PNC-associated protein PTB, its synthesis was inhibited by siRNA. This resulted in a reduction of the number of PNC-containing cells and the PNC size. Together, these findings suggest, as a working model, that PNCs may be involved in the metabolism of specific pol III transcripts in the transformed state and that PTB is one of the key elements mediating this process.

The redundancy of the mammalian heterochromatic compartment.

Two chromatin compartments are present in most mammalian cells; the first contains primarily euchromatic, early replicating chromatin and the second, primarily late-replicating heterochromatin, which is the subject of this review. Heterochromatin is concentrated in three intranuclear regions: the nuclear periphery, the perinucleolar space and in pericentromeric bodies. We review recent evidence demonstrating that the heterochromatic compartment is critically involved in global nuclear organization and the maintenance of genome stability, and discuss models regarding how this compartment is formed and maintained. We also evaluate our understanding of how heterochromatic sequences (herein named heterochromatic associated regions (HADs)) might be tethered within these regions and review experiments that reveal the stochastic nature of individual HAD positioning within the compartment. These investigations suggest a substantial level of functional redundancy within the heterochromatic compartment.

A mRNA and cognate microRNAs localize in the nucleolus.

We previously discovered that a set of 5 microRNAs are concentrated in the nucleolus of rat myoblasts. We now report that several mRNAs are also localized in the nucleoli of these cells as determined by microarray analysis of RNA from purified nucleoli. Among the most abundant of these nucleolus-localized mRNAs is that encoding insulin-like growth factor 2 (IGF2), a regulator of myoblast proliferation and differentiation. The presence of IGF2 mRNA in nucleoli was confirmed by fluorescence in situ hybridization, and RT-PCR experiments demonstrated that these nucleolar transcripts are spliced, thus arriving from the nucleoplasm. Bioinformatics analysis predicted canonically structured, highly thermodynamically stable interactions between IGF2 mRNA and all 5 of the nucleolus-localized microRNAs. These results raise the possibility that the nucleolus is a staging site for setting up particular mRNA-microRNA interactions prior to export to the cytoplasm.

Nucleolar tethering mediates pairing between the IgH and Myc loci.

Gene loci on different chromosomes can preferentially colocalize in the cell nucleus. However, many of the mechanisms mediating this spatial proximity remain to be elucidated. The IgH locus on Chromosome 12 and the Myc locus on Chromosome 15 are a well-studied model for gene colocalization in murine B cells, where the two loci are positioned in close proximity at a higher than expected frequency. These gene loci are also partners in the chromosomal translocation that causes murine plasmacytoma and Burkitt's lymphoma. Because both Chromosome 12 and Chromosome 15 carry nucleolar organizer regions (NORs) in the most commonly studied mouse strains, we hypothesized that NOR-mediated tethering of the IgH and Myc loci to shared nucleoli could serve as a mechanism to drive IgH:Myc colocalization. Using mouse strains that naturally carry nucleolar organizer regions (NORs) on different sets of chromosomes, we establish that IgH and Myc are positioned proximal to nucleoli in a NOR dependent manner and show that their joint association with nucleoli significantly increases the frequency of IgH and Myc pairing. Thus we demonstrate that simple nucleolar tethering can increase the colocalization frequency of genes on NOR-bearing chromosomes.

Tracking nuclear poly(A) RNA movement within and among speckle nuclear bodies and the surrounding nucleoplasm.

The movement of polyadenylated RNA transcripts (poly(A) RNA) through speckles in the nucleus can be detected and studied using fluorescence correlation microscopy (FCM) and photoactivation RNA tracking techniques. Speckles, sometimes called interchromatin granule clusters, are nuclear bodies that contain pre-mRNA splicing factors and poly(A) RNA. In the methods described here, speckles are marked in live cells using monomeric red fluorescent protein fused to SC35, a splicing protein that is a common speckle component. Endogenous poly(A) RNAs are tagged by in vivo hybridization with fluorescein-labeled oligo(dT) and FCM is performed at the marked speckles and in the nucleoplasm to measure the mobility of the tagged poly(A) RNA. The majority of the nuclear poly(A) RNA population diffuses rapidly throughout the nucleoplasm, and thus this method allows one to ask whether poly(A) RNA that is located in speckles at a given time is undergoing a dynamic transit or is, in contrast, a more immobile, perhaps structural, component. To visualize the movement of poly(A) RNA away from speckles, poly(A) RNA is tagged with caged-fluorescein-labeled oligo(dT) and speckle-associated poly(A) RNAs are specifically photoactivated using a laser beam directed through a pinhole in a rapid digital imaging microscopy system. The spatial distribution of the now-fluorescent RNA as it moves from the speckle photoactivation site is then recorded over time. Temperature and/or ATP levels can also be varied to test whether movement or localization of the poly(A) RNA is dependent on metabolic energy.

When untethered, something silent inside comes.

Heterochromatin usually is sequestered near the periphery and the nucleoli in mammalian nuclei. However, in terminally differentiated retinal rod cells of nocturnal mammals, heterochromatin instead accumulates in the interior, to give a so-called inside-out nuclear architecture. Solovei et al. now reports that in most cells, the lamin B receptor mediates peripheral localization early during development and that lamin A/C then takes over this tethering function during terminal differentiation. Furthermore, they show that the unique architecture of the nocturnal animal rod cell is caused by the absence of both tethers and can be phenocopied in LBR/lamin A/C double knockouts.

MicroRNA-206 colocalizes with ribosome-rich regions in both the nucleolus and cytoplasm of rat myogenic cells.

MicroRNAs are small, approximately 21- to 24-nt RNAs that have been found to regulate gene expression. miR-206 is a microRNA that is expressed at high levels in Drosophila, zebrafish, and mouse skeletal muscle and is thought to be involved in the attainment and/or maintenance of the differentiated state. We used locked nucleic acid probes for in situ hybridization analysis of the intracellular localization of miR-206 during differentiation of rat myogenic cells. Like most microRNAs, which are presumed to suppress translation of target mRNAs, we found that miR-206 occupies a cytoplasmic location in cultured myoblasts and differentiated myotubes and that its level increases in myotubes over the course of differentiation, consistent with previous findings in muscle tissue in vivo. However, to our surprise, we also observed miR-206 to be concentrated in nucleoli. A probe designed to be complementary to the precursor forms of miR-206 gave no nucleolar signal. We characterized the intracellular localization of miR-206 at higher spatial resolution and found that a substantial fraction colocalizes with 28S rRNA in both the cytoplasm and the nucleolus. miR-206 is not concentrated in either the fibrillar centers of the nucleolus or the dense fibrillar component, where ribosomal RNA transcription and early processing occur, but rather is localized in the granular component, the region of the nucleolus where final ribosome assembly takes place. These results suggest that miR-206 may associate both with nascent ribosomes in the nucleolus and with exported, functional ribosomes in the cytoplasm.

Photoactivation-based labeling and in vivo tracking of RNA molecules in the nucleus.

This protocol describes a method for observing and measuring the movement of RNA molecules in the nucleus of living mammalian cells. Caged fluorescein-labeled DNA oligonucleotides are introduced into living mammalian cells, where they demonstrably hybridize to complementary RNA. After site-specific photoactivation at desired sites within the cell, the RNA movements away from those sites are followed and digitally recorded using a rapid acquisition microscopy system.

Signal recognition particle assembly in relation to the function of amplified nucleoli of Xenopus oocytes.

The signal recognition particle (SRP) is a ribonucleoprotein machine that controls the translation and intracellular sorting of membrane and secreted proteins. The SRP contains a core RNA subunit with which six proteins are assembled. Recent work in both yeast and mammalian cells has identified the nucleolus as a possible initial site of SRP assembly. In the present study, SRP RNA and protein components were identified in the extrachromosomal, amplified nucleoli of Xenopus laevis oocytes. Fluorescent SRP RNA microinjected into the oocyte nucleus became specifically localized in the nucleoli, and endogenous SRP RNA was also detected in oocyte nucleoli by RNA in situ hybridization. An initial step in the assembly of SRP involves the binding of the SRP19 protein to SRP RNA. When green fluorescent protein (GFP)-tagged SRP19 protein was injected into the oocyte cytoplasm it was imported into the nucleus and became concentrated in the amplified nucleoli. After visiting the amplified nucleoli, GFP-tagged SRP19 protein was detected in the cytoplasm in a ribonucleoprotein complex, having a sedimentation coefficient characteristic of the SRP. These results suggest that the amplified nucleoli of Xenopus oocytes produce maternal stores not only of ribosomes, the classical product of nucleoli, but also of SRP, presumably as a global developmental strategy for stockpiling translational machinery for early embryogenesis.

Nuclear export of signal recognition particle RNA in mammalian cells.

In mammalian cells the signal recognition particle (SRP) consists of a approximately 300 nucleotide RNA and six proteins. Although the molecular structure and functional cycle of the SRP are both very well understood, far less is known about how the SRP is first assembled in the cell. Recent work has suggested that SRP assembly begins in the nucleoli. When NRK (rat fibroblast) cells were treated with leptomycin B (LMB), a specific inhibitor of the CRM1 nuclear export receptor, the level of SRP RNA increased in the nucleoli, as did the level of nucleolar 28S ribosomal RNA. Moreover, when a hamster cell line carrying a temperature-sensitive mutation in the guanine nucleotide exchange factor of the GTPase Ran (Ran-GEF) was shifted to the non-permissive temperature, the nucleolar level of SRP RNA increased. These results indicate that the steady-state concentration of SRP RNA in the nucleolus is sensitive to perturbations in nuclear import/export pathways.

Genome function and nuclear architecture: from gene expression to nanoscience.

Biophysical, chemical, and nanoscience approaches to the study of nuclear structure and activity have been developing recently and hold considerable promise. A selection of fundamental problems in genome organization and function are reviewed and discussed in the context of these new perspectives and approaches. Advancing these concepts will require coordinated networks of physicists, chemists, and materials scientists collaborating with cell, developmental, and genome biologists.