BBA - Molecular Cell Research (v.1783, #11)

Functional architecture of the cell nucleus by M. Lienhard Schmitz; Harald Herrmann (2041-2043).

Visualizing chromatin dynamics in intact cells by Ty C. Voss; Gordon L. Hager (2044-2051).
Chromatin and associated regulatory proteins regulate gene expression in the natural environment of the intact cell nucleus. Specific combinations of DNA-binding transcription factors and recruited coregulatory proteins alter the conformation of chromatin at promoters and enhancers of target genes to stimulate or repress transcription. The dynamic nature of the regulatory proteins active in these processes allows the cell to modulate gene expression very rapidly, an important feature in many physiological processes. Live cell imaging and photobleaching studies of fluorescently-tagged proteins reveal that many transcription factors and other chromatin-associated proteins rapidly move through the nucleoplasm. Transcription factors also transiently interact with specific regulatory sequences in chromatin, suggesting that gene activation does not require the formation of stable long-lived regulatory complexes on the chromatin. In this review we discuss how dynamic interactions allow transcriptional regulatory proteins find their targets within the nucleus, alter target chromatin structure, and modulate physiological gene expression.
Keywords: Chromatin; Nuclear receptor; Dynamics; Living cells; Hit-and-run; Transcription;

Eukaryotic cells store their genome inside a nucleus, a dedicated organelle shielded by a double lipid membrane. Pores in these membranes allow the exchange of molecules between the nucleus and cytoplasm. Inside the mammalian cell nucleus, roughly 2 m of DNA, divided over several tens of chromosomes is packed. In addition, protein and RNA molecules functioning in DNA-metabolic processes such as transcription, replication, repair and the processing of RNA fill the nuclear space. While many of the nuclear proteins freely diffuse and display a more or less homogeneous distribution across the nuclear interior, some appear to preferentially cluster and form foci or bodies. A non-random structure is also observed for DNA: increasing evidence shows that selected parts of the genome preferentially contact each other, sometimes even at specific sites in the nucleus. Currently a lot of research is dedicated to understanding the functional significance of nuclear architecture, in particular with respect to the regulation of gene expression. Here we will evaluate evidence implying that the folding of DNA is important for transcriptional control in mammals and we will discuss novel high-throughput techniques expected to further boost our knowledge on nuclear organisation.
Keywords: Nuclear organisation; Chromatin; Transcription; FISH; DNA structure;

Genome organization: Balancing stability and plasticity by Malte Wachsmuth; Maïwen Caudron-Herger; Karsten Rippe (2061-2079).
The cell needs to stably maintain its genome and protect it from uncontrolled modifications that would compromise its function. At the same time, the genome has to be a plastic structure that can dynamically (re)organize to allow the cell to adopt different functional states. These dynamics occur on the nanometer to micrometer length scale, i.e. ranging from the level of single proteins up to that of whole chromosomes, and on a microsecond to hour time scale. Here, we review different contributions to the dynamic features of the genome, describe how they are determined experimentally, and discuss the results of these measurements in terms of how the requirements for stability and plasticity are accommodated with specific activities in the nucleus.
Keywords: Genome organization; Nuclear structure; Fluorescence microscopy; Macromolecular dynamics and interactions; Diffusion;

Functional architecture of the cell nucleus: Towards comprehensive toponome reference maps of apoptosis by Walter Schubert; Manuela Friedenberger; Marcus Bode; Andreas Krusche; Reyk Hillert (2080-2088).
We have recently described the MELC/TIS fluorescence robot technology that is capable of colocalizing at least a hundred different molecular cell components in one cell. The technology reveals new hierarchical properties of protein network organisation, referred to as the toponome, in which topologically confined protein clusters are interlocked within the structural framework of the cell. In this study we have applied MELC/TIS to construct a three-dimensional toponome map of the cell nucleus of a single human hepatocyte undergoing apoptosis. The map reveals six different spatially separated toponome domains in the nuclear interior of one apoptotic cell. In the drive to decipher the apoptosis-specific molecular network on the single cell level, the present toponome map is a first milestone towards the construction of much larger maps addressing hundreds of molecular cell components across the stages of apoptosis.
Keywords: Apoptosis; Toponome; TIS; Cell nucleus; Fluorescence microscopy; Liver cell;

Structural analysis of interphase X-chromatin based on statistical shape theory by Siwei Yang; Doris Illner; Kathrin Teller; Irina Solovei; Roel van Driel; Boris Joffe; Thomas Cremer; Roland Eils; Karl Rohr (2089-2099).
The 3D folding structure formed by different genomic regions of a chromosome is still poorly understood. So far, only relatively simple geometric features, like distances and angles between different genomic regions, have been evaluated. This work is concerned with more complex geometric properties, i.e., the complete shape formed by genomic regions. Our work is based on statistical shape theory and we use different approaches to analyze the considered structures, e.g., shape uniformity test, 3D point-based registration, Fisher distribution, and 3D non-rigid image registration for shape normalization. We have applied these approaches to analyze 3D microscopy images of the X-chromosome where four consecutive genomic regions (BACs) have been simultaneously labeled by multicolor FISH. We have acquired two sets of four consecutive genomic regions with an overlap of three regions. From the experimental results, it turned out that for all data sets the complete structure is non-random. In addition, we found that the shapes of active and inactive X-chromosomal genomic regions are statistically independent. Moreover, we reconstructed the average 3D structure of chromatin in a small genomic region (below 4 Mb) based on five BACs resulting from two overlapping four BAC regions. We found that geometric normalization with respect to the nucleus shape based on non-rigid image registration has a significant influence on the location of the genomic regions.
Keywords: Statistical shape theory; Geometrical morphometrics; Shape distribution; 3D chromatin structure; Genomic region; 3D multicolor FISH microscopy; Non-rigid image registration; Cell nuclei; Shape normalization;

Macromolecular crowding and its potential impact on nuclear function by Karsten Richter; Michelle Nessling; Peter Lichter (2100-2107).
It is well established, that biochemical reactions are dependent on pH, ionic strength, temperature and the concentration of reactants. However, the steric repulsion among bulky components of biological systems also affect biochemical behavior: The ‘excluded volume effect of macromolecular crowding’ drives bulky components into structurally compact organizations, increases their thermodynamic activities and slows down diffusion. The very special composition of the cell nucleus, which is packed with high-molecular chromatin, ribonucleo-particles and associated proteins, suggests that crowding-effects are part of nuclear functionality. Realizing that many nuclear processes, notably gene transcription, hnRNA splicing and DNA replication, use macromolecular machines, and taking into account that macromolecular crowding provides a cooperative momentum for the assembly of macromolecular complexes, we here elaborate why macromolecular crowding may be functionally important in supporting the statistical significance of nuclear activities.
Keywords: Heterochromatin; Microcompartmentalization; Noise in gene expression; Nuclear architecture; Phase separation;

The Cajal body by Glenn E. Morris (2108-2115).
The Cajal body, originally identified over 100 years ago as a nucleolar accessory body in neurons, has come to be identified with nucleoplasmic structures, often quite tiny, that contain coiled threads of the marker protein, coilin. The interaction of coilin with other proteins appears to increase the efficiency of several nuclear processes by concentrating their components in the Cajal body. The best-known of these processes is the modification and assembly of U snRNPs, some of which eventually form the RNA splicing machinery, or spliceosome. Over the last 10 years, research into the function of Cajal bodies has been greatly stimulated by the discovery that SMN, the protein deficient in the inherited neuromuscular disease, spinal muscular atrophy, is a Cajal body component and has an essential role in the assembly of spliceosomal U snRNPs in the cytoplasm and their delivery to the Cajal body in the nucleus.
Keywords: Cajal body; Coiled body; Gems; Coilin; SMN; Spinal muscular atrophy; RNA splicing; Spliceosome; snRNP; Methylase; Methylosome; Gemin2; Gemin3; Gemin4; Gemin5; Gemin6; Gemin7; Gemin8; Unrip; Sm protein; snoRNA; scaRNA; Pseudouridylation; 3'-end processing; Telomerase; miRNA; PML body; Fibrillarin;

Pseudo-NORs: A novel model for studying nucleoli by José-Luis Prieto; Brian McStay (2116-2123).
Nucleolar organiser regions (NORs) are comprised of tandem arrays of ribosomal gene (rDNA) repeats that are transcribed by RNA polymerase I (Pol I), ultimately resulting in formation of a nucleolus. Upstream binding factor (UBF), a DNA binding protein and component of the Pol I transcription machinery, binds extensively across the rDNA repeat in vivo. Pseudo-NORs are tandem arrays of a heterologous DNA sequence with high affinity for UBF introduced into human chromosomes. In this review we describe how analysis of pseudo-NORs has provided important insights into nucleolar formation. Pseudo-NORs mimic endogenous NORs in a number of important respects. On metaphase chromosomes both appear as secondary constrictions comprised of undercondensed chromatin. The transcriptional silence of pseudo-NORs provides a platform for studying the transcription independent recruitment of factors required for nucleolar formation by this specialised chromatin structure. During interphase, pseudo-NORs appear as distinct and novel sub-nuclear bodies. Analysis of these bodies and comparison to their endogenous counterpart has provided insights into nucleolar formation and structure.
Keywords: Nucleolus; Nucleolar organiser region; Ribosomal gene; UBF; Pseudo-NOR;

HIPKs: Jack of all trades in basic nuclear activities by Cinzia Rinaldo; Francesca Siepi; Andrea Prodosmo; Silvia Soddu (2124-2129).
Over the past decade several investigators have reported on the physical interaction of serine/threonine kinases of the homeodomain interacting-protein family (HIPKs) with increasing number of nuclear factors and on their localization in different nuclear sub-compartments. Although we are still far from a global understanding of the molecular consequences of HIPK subnuclear compartmentalization, the spatial description of particular interactions and posttranslational modifications promoted by these kinases on key cellular regulators might provide relevant insights. Here we will discuss the possible implications of the HIPK subnuclear localization in the regulation of gene transcription and in the cell response to stress.
Keywords: HIPK; HIPK domain; PML-body; p53; Pc2; DNA-damage response;

The single-celled ciliate Tetrahymena thermophila possesses two versions of its genome, one germline, one somatic, contained within functionally distinct nuclei (called the micronucleus and macronucleus, respectively). These two genomes differentiate from identical zygotic copies. The development of the somatic nucleus involves large-scale DNA rearrangements that eliminate 15 to 20 Mbp of their germline-derived DNA. The genomic regions excised are dispersed throughout the genome and are largely composed of repetitive sequences. These germline-limited sequences are targeted for removal from the genome by a RNA interference (RNAi)-related machinery that directs histone H3 lysine 9 and 27 methylation to their associated chromatin. The targeting small RNAs are generated in the micronucleus during meiosis and then compared against the parental macronucleus to further enrich for germline-limited sequences and ensure that only non-genic DNA segments are eliminated. Once the small RNAs direct these chromatin modifications, the DNA rearrangement machinery, including the chromodomain proteins Pdd1p and Pdd3p, assembles on these dispersed chromosomal sequences, which are then partitioned into nuclear foci where the excision events occur. This DNA rearrangement mechanism is Tetrahymena's equivalent to the silencing of repetitive sequences by the formation of heterochromatin. The dynamic nuclear reorganization that occurs offers an intriguing glimpse into mechanisms that shape nuclear architecture during eukaryotic development.
Keywords: Heterochromatin; RNAi; DNA rearrangement; Chromatin; Chromodomain; Genome surveillance;

Joining the dots: Production, processing and targeting of U snRNP to nuclear bodies by Debra J. Shaw; Paul Eggleton; Philip J. Young (2137-2144).
The spliceosome is the RNP complex than catalyses the removal of introns from the Uridine-rich small nuclear ribonucleoproteins (U snRNPs) that make up the main components of this complex. The production of these RNPs is an intricate process, involving several key stages. These include: 1) the transcription of the U snRNAs; 2) their nuclear export; 3) the cytoplasmic assembly of the U snRNP; 4) their nuclear import; 5) their processing within Cajal bodies and the nucleolus; and 6) their storage in interchromatin granule clusters (IGCs). This review focuses on each of these stages, discussing the key complexes involved as well as the trafficking and targeting mechanisms involved.
Keywords: U snRNP; Nuclear export; Sm core proteins; Nuclear import sequences; Survival motor neuron (SMN); Cajal bodies; Nucleoli; Interchromatin granule clusters (IGC);

The promyelocytic leukemia protein PML and its associated nuclear bodies are hot topics of investigation. This interest arises for multiple reasons including the tight link between the integrity of PML nuclear bodies and several disease states and the impact of the PML protein and PML nuclear bodies on proliferation, apoptosis and viral infection. Unfortunately, an understanding of the molecular underpinnings of PML nuclear body function remains elusive. Here, a general overview of the PML field is provided and is extended to discuss whether some of the basic tenets of “PML-ology” are still valid. For instance, recent findings suggest that some components of PML nuclear bodies form bodies in the absence of the PML protein. Also, a new model for PML nuclear body function is proposed which provides a unifying framework for its effects on diverse biochemical pathways such as Akt signaling and the p53-Mdm2 axis. In this model, the PML protein acts as an inhibitor of gene expression post-transcriptionally via inhibiting a network node in the eIF4E RNA regulon. An example is given for how the PML RNA regulon model provided the basis for the development of a new anti-cancer strategy being tested in the clinic.
Keywords: PML; ND10; POD; eIF4E; RNA regulon/operon;

Homologous chromosomes can pair in somatic and germ line cells, and many mechanisms have been proposed to explain how they do so. One popular class of models involves base-pairing between DNA strands catalyzed by recombination proteins, but pairing still occurs in mutants lacking the relevant functional proteins. We discuss an alternative based on two observations: transcription occurs in factories that specialize in transcribing specific gene sub-sets, and chromosomes only pair when transcribed. Each chromosome in the haploid set has a unique array of transcription units strung along its length; we suggest each is organized into clouds of loops tethered to specialized factories. Only homologs share similar strings of clouds and factories. Pairing begins when a promoter on one chromosome initiates in the homologous and specialized factory organized mainly by its homologous partner. This transiently ties the two homologs together, to increase the chances that adjacent promoters initiate in their homologous factories and that the two homologs will be zipped together. Then, interactions between promoters and RNA polymerases in the factories mediate pairing.
Keywords: Chromosome pairing; Homology search; Mieosis; Recombination; Transcription factory;

Long range chromatin interactions involved in gene regulation by Marek Bartkuhn; Rainer Renkawitz (2161-2166).
Long-distance chromatin interaction has been proposed and demonstrated for enhancer elements separated from the gene by hundreds or thousands of base pairs. This paved the way for the detection of additional enhancer properties, such as the regulation of interaction, and the contacting of genes in trans on other chromosomes. The outspread arrangement of regulatory elements and transcription units requires insulators to prevent the functional interference of enhancer elements with inappropriate promoters. Apparently, insulators mediate differential chromatin folding to allow or to prevent enhancers from contacting specific promoters. The factor CTCF is often involved in bridging separated chromatin regions. In addition to interchromosomal contacts, intrachromosomal interactions have been demonstrated for genes with a similar regulation, such as active genes, estrogen induced genes and imprinted genes. With more sophisticated and sensitive methods combined with deep sequencing and array technology, a huge number of long range interactions can expected to be characterized in the near future.
Keywords: Chromatin; Three-dimensional interaction; Enhancer; Imprinting; CTCF;

The cell biology of DNA methylation in mammals by Egor Prokhortchouk; Pierre-Antoine Defossez (2167-2173).
In this review, we will provide a brief reminder of epigenetic phenomena in general, and DNA methylation in particular. We will then underline the characteristics of the in vivo organization of the genome that limit the applicability of in vitro results. We will use several examples to point out the connections between DNA methylation and nuclear architecture. Finally, we will outline some of the hopes and challenges for future research in the field. The study of DNA methylation, its effectors, and its roles, illustrates the complementarity of in vitro approaches and cell biology.
Keywords: Epigenetics; DNA methylation; Cell biology; Mammals;

Nuclear architecture and gene regulation by Elena Fedorova; Daniele Zink (2174-2184).
The spatial organization of eukaryotic genomes in the cell nucleus is linked to their transcriptional regulation. In mammals, on which this review will focus, transcription-related chromatin positioning is regulated at the level of chromosomal sub-domains and individual genes. Most of the chromatin remains stably positioned during interphase. However, some loci display dynamic relocalizations upon transcriptional activation, which are dependent on nuclear actin and myosin. Transcription factors in association with chromatin modifying complexes seem to play a central role in regulating chromatin dynamics and positioning. Recent results obtained in this regard also give insight into the question how the different levels of transcriptional regulation are integrated and coordinated with other processes involved in gene expression. Corresponding findings will be discussed.
Keywords: Nuclear architecture; Chromatin positioning; Gene regulation; Transcription factor; Chromatin dynamics; Histone modifications;

Role of nuclear bodies in apoptosis signalling by Eva Krieghoff-Henning; Thomas G. Hofmann (2185-2194).
Promyelocytic leukemia nuclear bodies (PML NBs) are dynamic macromolecular multiprotein complexes that recruit and release a plethora of proteins. A considerable number of PML NB components play vital roles in apoptosis, senescence regulation and tumour suppression. The molecular basis by which PML NBs control these cellular responses is still just beginning to be understood. In addition to PML itself, numerous further tumour suppressors including transcriptional regulator p53, acetyl transferase CBP (CREB binding protein) and protein kinase HIPK2 (homeodomain interacting protein kinase 2) are recruited to PML NBs in response to genotoxic stress or oncogenic transformation and drive the senescence and apoptosis response by regulating p53 activity. Moreover, in response to death-receptor activation, PML NBs may act as nuclear depots that release apoptotic factors, such as the FLASH (FLICE-associated huge) protein, to amplify the death signal. PML NBs are also associated with other nuclear domains including Cajal bodies and nucleoli and share apoptotic regulators with these domains, implying crosstalk between NBs in apoptosis regulation. In conclusion, PML NBs appear to regulate cell death decisions through different, pathway-specific molecular mechanisms.
Keywords: Apoptosis; Nuclear domain; PML nuclear body; Genotoxic stress; p53; Death-receptor signalling; Crosstalk;

Nuclear bodies in neurodegenerative disease by John Woulfe (2195-2206).
Neurodegenerative diseases are characterized by a relentlessly progressive loss of the functional and structural integrity of the central nervous system. In many cases, these diseases arise sporadically and the causes are unknown. The abnormal aggregation of protein within the cytoplasm or the nucleus of brain cells represents a unifying pathological feature of these diseases. There is increasing evidence for nuclear dysfunction in neurodegenerative diseases. How this relates to protein aggregation in the context of “cause and effect” remains to be determined in most cases. Co-ordinated nuclear function is predicated on the activity of distinct nuclear subdomains, or nuclear bodies, each responsible for a specific function. If nuclear dysfunction represents an important etiopathological feature in neurodegenerative disease, then this should be reflected by functional and/or morphological alterations in this nuclear compartmentalization. For most neurodegenerative diseases, evidence for nuclear dysfunction, with attendant consequences for nuclear architecture, is only beginning to emerge. In this review, I will discuss neurodegenerative diseases in the context of nuclear dysfunction and, more specifically, alterations in nuclear bodies. Although research in this field is in its infancy, identifying alterations in the nucleus in neurodegenerative disease has potentially profound implications for elucidating the pathogenesis of these disorders.
Keywords: Neurodegenerative disease; Nuclear inclusion; Nuclear body; Frontotemporal dementia; Spinal muscular atrophy; Alzheimer's disease;

Nuclear domains 10 (ND10), alternatively termed PML nuclear bodies (PML-NBs) or PML oncogenic domains (PODs), have been discovered approximately 15 years ago as a nuclear substructure that is targeted by a variety of viruses belonging to different viral families. This review will summarize the most important structural and functional characteristics of ND10 and its major protein constituents followed by a discussion of the current view regarding the role of this subnuclear structure for various DNA and RNA viruses with an emphasis on herpesviruses. It is concluded that accumulating evidence argues for an involvement of ND10 in host antiviral defenses either via mediating an intrinsic immune response against specific viruses or via acting as a component of the cellular interferon pathway.
Keywords: Nuclear domain 10; PML-bodies; PML; Sp100; hDaxx; Antiviral defense; Intrinsic immunity; Interferon;