The EMBO workshop on “Chromatin organization, structure and dynamics”; organized by Ivan Raška; Roland Foisner and Yosef Gruenbaum, was held in Prague, 9–13 April 2011.
Chromatin structure, organization and dynamics underlie every aspect of genome function. Over the past few years, the combined use of novel tools, for instance superresolution light microscopy and high throughput sequencing, with structural, cell biological, biochemical and genetic analyses, have led to major progress in our understanding of the relationships between chromatin structure and its functions in replication, transcription and RNA processing, and mitosis. Importantly, the progress in basic research in this field has also found increasing application in human medicine.
In the workshop, oral and poster presentations by students and postdoctoral fellows were complemented by talks from leading researchers in the field. The keynote address was given by Joseph Gall, a pioneer of chromatin structure, nuclear organization and RNA biology. Gall’s talk set the stage for the subsequent days of presentations, focusing on the complexity of nuclear organization and highlighting the potential for new discovery through innovative application of advanced techniques. The lectures by the invited speakers and by speakers selected from submitted abstracts were organized in eight sessions and formed the core of the workshop. The session topics covered nuclear organization and chromatin structure, epigenetic regulation of chromatin, chromatin behavior during mitosis, chromatin and the nuclear envelope, nuclear pore complexes and their roles in transport and gene activity, chromatin organization and transcription, DNA replication and repair, and aspects of RNA processing. Excitingly, several contributions had a direct impact on medical science, such as cancer biology and laminopathies.
Workshop participants enjoyed excellent science. The selection of participants representing a vast palette of chromatin investigators and topics as well as the friendly atmosphere of the meeting allowed numerous personal contacts and exchange of ideas. Hence, the workshop promoted new ways of analyzing and deciphering complex biological processes associated with chromatin.
To promote the progress in the field reported on in this workshop, Roland Foisner, the Editor-in-Chief of Nucleus, invited meeting participants to submit research articles and reviews to a special issue of Nucleus on “Chromatin organization, structure and dynamics”. As a result, 17 workshop participants contributed, covering many aspects of scientific discoveries discussed during the EMBO workshop.
Yosef Gruenbaum Harald Herrmann Karla Neugebauer
Guest Associate Editors
Defined as a chromatin structure that remains condensed throughout the cell cycle heterochromatin is generally transcriptionally silent and is characterized by a specific molecular signature. Constitutive heterochromatin at the pericentromere is a conserved feature throughout evolution, which impacts genome stability. Here, we will summarize recent advances in our understanding of the dynamics of mouse pericentric heterochromatin during the cell cycle and development. Comparison with heterochromatin maintenance in fission yeast will enable discussions of the common basic principles and various mechanisms exploited in the distinct organisms
In the past 15 years our perception of nuclear envelope function has evolved perhaps nearly as much as the nuclear envelope itself evolved in the last 3 billion years. Historically viewed as little more than a diffusion barrier between the cytoplasm and the nucleoplasm, the nuclear envelope is now known to have roles in the cell cycle, cytoskeletal stability and cell migration, genome architecture, epigenetics, regulation of transcription, splicing, and DNA replication. Here we will review both what is known and what is speculated about the role of the nuclear envelope in genome organization, particularly with respect to the positioning and repositioning of genes and chromosomes within the nucleus during differentiation.
The nuclear lamina is a protein-rich network located directly underneath the inner nuclear membrane of metazoan nuclei. The components of the nuclear lamina have been implicated in nearly all nuclear functions; therefore, understanding the structural, mechanical, and signal transducing properties of these proteins is crucial. In addition, mutations in many of these proteins cause a wide range of human diseases, the laminopathies. The structure, function, and interaction of the lamina proteins are conserved among metazoans, emphasizing their fundamental roles in the nucleus. Several of the advances in the field of the nuclear lamina have come from studies performed in Caenorhabditis elegans or on C. elegans proteins expressed in vitro. Here, we discuss the current knowledge about the nuclear lamina, including an overview of the technical tools offered by C. elegans that make it a powerful model organism for the study of the nuclear lamina and laminopathic diseases.
Eukaryotic chromosomes are condensed into several hierarchical levels of complexity: DNA is wrapped around core histones to form nucleosomes, nucleosomes form a higher-order structure called chromatin, and chromatin is subsequently compartmentalized in part by the combination of multiple specific or unspecific long-range contacts. The conformation of chromatin at these three levels greatly influences DNA metabolism and transcription. One class of chromatin regulatory proteins called insulator factors may organize chromatin both locally, by setting up barriers between heterochromatin and euchromatin, and globally by establishing platforms for long-range interactions. Here, we review recent data revealing a global role of insulator proteins in the regulation of transcription through the formation of clusters of long-range interactions that impact different levels of chromatin organization.
Every time a cell divides it must ensure that its genetic information is accurately duplicated and dis-tributed equally to the two daughter cells. This fundamental biological process is conserved through-out all kingdoms of life and relies on the correct and complete duplication of the DNA before a cell can divide and give rise to other cells or to multicellular organisms. Any mistakes in this process can result in genetic mutations or karyotype aberrations, which may lead to disease or even death. Whereas in prokaryotes the entire genome is replicated from a single origin, the increased genome size and complexity in mammals requires the spatio-temporal coordination of thousands of replica-tion origins. Furthermore, this spatio-temporal order of genome replication changes throughout de-velopment and cellular differentiation. Here we present and discuss current knowledge on the con-trol of DNA replication dynamics in mammals and the role of chromatin modifications in this basic biological process.
In most organisms, telomeres are extended by telomerase and contain GC-rich repeats. Drosophila telomeres are elongated by occasional transposition of specialized retroelements rather than telomerase activity, and are assembled independently of the sequence of the DNA termini. Recent work has shown that Drosophila telomeres are capped by a complex, we call terminin, which includes HOAP, HipHop, Moi and Ver; these are fast-evolving proteins that prevent telomere fusion, directly interact with each other, and appear to localize and function only at telomeres. With the possible exception of Ver that contains an OB fold domain structurally similar to the Stn1 OB fold, none of the terminin proteins is evolutionarily conserved outside the Drosophila species. Human telomeres are protected by the shelterin complex, which comprises six proteins that bind chromosome ends in a sequence-dependent manner. Shelterin subunits are not fast-evolving proteins and are not conserved in flies, but localize and function only at telomeres like the terminin components. Based on these findings, we propose that concomitant with telomerase loss Drosophila rapidly evolved terminin to bind chromosome ends in a sequence-independent fashion, and that terminin is functionally analogous to shelterin.
DNA methylation plays a central role in the epigenetic regulation of gene expression during development and disease. Remarkably, the complex and changing patterns of genomic DNA methylation are established and maintained by only three DNA methyltransferases. Here we focus on DNMT1, the major and ubiquitously expressed DNA methyltransferase in vertebrates, to outline possible regulatory mechanisms. A list of all protein interactions and post-translational modifications reported for DNMT1 clearly shows that DNMT1, and by extension also DNA methylation in general, are functionally linked with several other epigenetic pathways and cellular processes. General themes of these interactions and modifications include the activation, stabilization and recruitment of DNMT1 at specific sites and heterochromatin regions. For a comprehensive understanding of the regulation of DNA methylation it is now necessary to systematically quantify the interactions and modifications of DNMT1, to elucidate their function at the molecular level and to integrate these data at the cellular level.
When living egg chambers of Drosophila are isolated in a saline solution and gently squashed between a microscope slide and coverslip, prominent nuclear bodies (1 - 20 mm diameter) can be seen inside the oocyte nucleus or germinal vesicle (GV). These bodies do not pre-exist within the GV and are not seen in material that is fixed in paraformaldehyde before squashing. Instead, they form spontaneously within minutes after an egg chamber is damaged and the cytoplasm is exposed to the isolation medium. Electron microscopy shows that the bodies lack an investing membrane and consist of closely packed, irregular particles 30-50 nm in diameter. We used GFP-tagged proteins from the Carnegie Protein Trap Library to identify 22 proteins that are either enriched in the bodies or excluded from them. We were unable to discern common features of proteins that are concentrated in the bodies, such as isoelectric point, molecular weight, or biological process. Induced bodies are formed in GVs of flies that are null for coilin or WDR79, proteins that are required for formation of Cajal bodies (CBs). We performed fluorescence recovery after photobleaching (FRAP) experiments on five GFP-tagged proteins that are enriched in the bodies. Four of the proteins regained the full pre-bleach fluorescence intensity, indicating that the contents of the bodies are in dynamic equilibrium with the surrounding nucleoplasm. Induced nuclear bodies presumably form as a result of unusual physico-chemical changes in the Drosophila GV. We suggest that their behavior serves as a useful model for self-assembly of nuclear bodies in general, and we discuss the possibility that similar bodies may occur normally in cells of other organisms.
The multi-layered organization of the genome in a large nucleoprotein complex termed chromatin regulates nuclear functions by establishing subcompartments with distinct DNA-associated activities. Here, we demonstrate that RNA plays an important role in maintaining a decondensed and biologically active interphase chromatin conformation in human and mouse cell lines. As shown by RNase A microinjection and fluorescence microscopy imaging, digestion of single-stranded RNAs induced a distinct micrometer scale chromatin aggregation of these decondensed regions. In contrast, pericentric heterochromatin was more resistant to RNase A treatment. We identified a class of coding RNA transcripts that are responsible for this activity, and thus termed these ‘chromatin-interlinking’ RNAs or ciRNAs. The initial chromatin distribution could be restored after RNase A treatment with a purified nuclear RNA fraction that was analyzed by high-throughput sequencing. It comprised long >500 nucleotides (nt) RNA polymerase II (RNAP II) transcripts that were spliced, depleted of polyadenylation and was enriched with long 3'-untranslated regions (3’-UTRs) above ~800 nt in length. Furthermore, similar reversible changes of the chromatin conformation and the RNAP II distribution were induced by either RNA depletion or RNAP II inhibition. Based on these results we propose that ciRNAs could act as genome organizing architectural factors of actively transcribed chromatin compartments.
We have investigated and quantified the nuclear A-type lamin pool from human HeLa S3 suspension cells with respect to their distribution to detergent soluble and insoluble fractions. We devised a sequential extraction protocol and found that maximally 10% of A-type lamins are recovered in the soluble fraction. Notably, lamin C is enriched in low detergent fractions and only with 0.5% Nonidet P-40 lamin A and C are recovered in ratios nearly equivalent to those found in whole cell extracts and in the lamina fraction. Authentic nucleoplasmic proteins such as LAP2a, pRB and p53 are co-extracted to a large part together with the A-type lamins in these fractions. By sucrose density centrifugation we revealed that the majority of lamins co-sedimented with human IgG indicating they form rather small complexes in the range of dimers and slightly larger complexes. Some lamin A - but not lamin C - is obtained in addition in a much faster sedimenting fraction. Authentic nuclear proteins such as PCNA, p53 and LAP2a were found both in the light and the heavy sucrose fractions together with lamin A. Last but not least, immunoprecipitation experiments from both soluble fractions and from RIPA lysates of whole cells revealed that lamin A and lamin C do not form heterodimers but segregate practically completely. Correspondingly, immunofluorescence microscopy of formaldehyde-fixed cells clearly demonstrated that lamin A and C are localized at least in part to distinct patches within the lamina. Hence, the structural segregation of lamin A and C is indeed retained in the nuclear envelope to some extent too.
Up-regulated expression of lamin A has been implicated in increased cell invasiveness and mortality in colorectal cancer. Here we use quantitative proteomics to investigate lamin A regulated changes in the cytoskeleton that might underpin increased cell motility. Using siRNA knockdown of lamin A in a model cell line (SW480/lamA) we confirm that the presence of lamin A promotes cell motility. Using an enhanced technique to prepare cytoskeleton fractions in combination with 2D DiGE we were able to accurately and reproducibly detect changes in the representation of protein species within the cytoskeleton as low as 20%. In total 64 protein spots displayed either increased or decreased representation within the cytoskeleton of SW480/lamA cells compared to controls. Of these the identities of 29 spots were determined by mass spectrometry. A majority were multiple forms of three classes of proteins, including components of the actin and IF cytoskeletons, protein chaperones and translation initiation and elongation factors. In particular our data reveal that the representation of tissue transglutaminase 2, which is known to modify elements of the cytoskeleton and is associated with cancer progression, was highly over-represented in the cytoskeleton fraction of SW480/lamA cells. Overall, our data are consistent with changed protein cross-linking and folding that favours the formation of dynamic actin filaments over stress fibres accounting for the altered cell motility properties in SW480/lamA cells.
Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease caused by a polyglutamine expansion in ataxin-7, a subunit of the SAGA coactivator, which leads to progressive neuronal dysfunction and cell death in cerebellum, brainstem and retina. Increased nuclear volume, chromatin decondensation and deregulated gene expression were reported in a SCA7 mouse model expressing mutant ataxin-7 in rod photoreceptors. We analyzed the SCA7-induced chromatin reorganization by immunogold labeling, stereology, electron tomography and showed that in SCA7 rods the most external heterochromatin ring, corresponding to facultative heterochromatin, becomes fragmented and decondensed. The amounts of acetylated histone H3 and H4 tails were found to be unchanged in nuclear extracts of SCA7 retinas and their cellular distribution appeared similar in wild-type and SCA7 mice in so far that in both cases acetylated histones are positioned at the interface between eu- and hetero-chromatin. We found that the amount of the linker histone H1c is strongly reduced in nuclear extracts of SCA7 retinas and that the cellular distribution of H1c is particularly altered in the facultative heterochromatin compartment. The decreased histone H1c content thus provides a coherent explanation for the chromatin decondensation observed in SCA7 rod photoreceptor nuclei.
Pre-replication complexes (pre-RCs) are assembled onto DNA during late mitosis and G1 to license replication origins for use in S phase. In order to prevent re-replication of DNA, licensing must be completely shutdown prior to entry into S phase. While mechanisms preventing re-replication during S phase and mitosis have been elucidated, the means by which cells first prevent licensing during late G1 phase are poorly understood. We have employed a hybrid mammalian / Xenopus egg extract replication system to dissect activities that inhibit replication licensing at different stages of the cell cycle in Chinese Hamster Ovary (CHO) cells. We find that soluble extracts from mitotic cells inhibit licensing through a combination of geminin and Cdk activities, while extracts from S-phase cells inhibit licensing predominantly through geminin alone. Surprisingly however, geminin did not accumulate until after cells enter S phase. Unlike extracts from cells in early G1 phase, extracts from late G1 phase and early S phase cells contained an inhibitor of licensing that could not be accounted for by either geminin or Cdk. Moreover, inhibiting cyclin and geminin protein synthesis or inhibiting Cdk activity early in G1 phase did not prevent the appearance of inhibitory activity. These results suggest that a soluble inhibitor of replication licensing appears prior to entry into S phase that is distinct from either geminin or Cdk activity. Our hybrid system should permit the identification of this and other novel cell cycle regulatory activities.
The active and inactive X (Xa;Xi) territory with its seemingly highly compacted Barr body in nuclei of female mammalian cells provide a key example for studies of structure/function relationships in homologous chromosomes with different functional properties. Here we used about 300 human X-specific large insert clones to generate probe sets, which target physically or functionally defined sub-chromosomal segments. We combined 3D multicolor FISH with quantitative 3D image analysis in order to compare the higher order organization in Xi-and Xa-territories in human diploid fibroblasts (HDFs) at various length scales ranging from about 50 Mb down to 1 Mb. Xi-territories were characterized by a rounder shape as compared to the flatter and more extended shape of Xa-territories. The overall compaction of the entire Xi-territory, including the Barr body, was only 1.2-fold higher than the Xa-territory. Significant differences, however, were noted between distinct subchromosomal segments: At 20 Mb length scales higher compaction in Xi-territories was restricted to specific segments, but higher compaction in these segments was not correlated with gene density, transcriptional activity, LINE content or histone markers locally enriched in Xi-territories. Notably, higher compaction in Xi-territories observed for 20 Mb segments was not reflected accordingly by inclosed segments of 1-4 Mb. We conclude that compaction differences result mainly from a regrouping of ~1 Mb chromatin domains rather than from an increased condensation of individual domains. In contrast to a previous report, genes subject to inactivation as well as escaping from inactivation were not excluded from the interior of the Barr body.
Changes in the nuclear structure and function during the cell cycle are thought to be correlated with lamins phosphorylation. Here, we report the identification of new in vivo phosphorylation sites on Drosophila melanogater lamin Dm using immunoisolation and mass spectrometry with collision-induced peptide fragmentation (Electrospray-Linear Trap Quadrupole- Fourier Transform Ion Cyclotron Resonance MS/MS). We identified S19 and confirmed previously suggested S595 as phosphorylated amino acid residues on embryonic lamin Dm. We also found that T597 is phosphorylated in vivo in cultured Kc cells while S595 in embryos, which suggests that different neighboring phosphoacceptors may be modified within the same region. We demonstrate also that Drosophila melanogaster lamin Dm in very early (syncytial) embryos is almost completely dispersed through the entire embryo. Only fraction of lamin Dm is associated with nuclei and nuclear envelopes. In later stages, due to the synchronization of mitosis, lamin Dm may be both nuclear and cytoplasmic in the same embryo. Our results provide a new and essential data for better understanding of the lamin phosporylation in development and cell cycle regulation in Drosophila.
At the onset of Drosophila metamorphosis the steroid hormone ecdysone induces a process leading to a rapid degeneration of the larval salivary glands (SGs). Ecdysone acts through the ecdysone receptor heterodimer, which activates primary response genes. In particular these genes include the Broad-Complex (BR-C) gene encoding a set of BTB/POZ-transcription factors, among which the Z1 isoform is critical for SG cell death. The timing of SG disappearance depends upon of p127l(2)gl, a cytoskeletal tumor suppressor that interacts with nonmuscle myosin II heavy chain (nmMHC) encoded by the zipper (zip) gene. Reduced l(2)gl expression delays SG histolysis whereas over-expression accelerates this process without affecting larval and pupal development. However, the mechanism by which l(2)gl controls SG histolysis remains yet unknown. Here we analyze the regulation controlled by p127l(2)gl and nmMHC in the cytoplasm on the association of BR-C Z1 with chromatin and remodeling factors, such as Rpd3, Sin3A, and Smrter. In wild-type SGs these factors bind to chromatin but in l(2)gl SGs they accumulate in the cytoplasm and the cortical nuclear zone (CNZ). Similar chromatin exclusion occurs in SGs of developmentally delayed zipE(br)/+ larvae or can be achieved by high levels of nmMHC synthesis. The present data show that p127l(2)gl and nmMHC regulate the access of BR-C Z1, Rpd3, Sin3A, and Smrter to chromatin. As the interaction between p127l(2)gl and nmMHC occurs in the cytoplasm, we propose that these nuclear factors are processed by p127l(2)gl and then released from p127l(2)gl by nmMHC to allow their binding to chromatin. This process may constitute a novel mechanism of gene regulation, which in the absence of p127l(2)gl, or excessive amounts of nmMHC, could lead to a fixed configuration in the pattern of gene expression that prevents further progression of SG differentiation, and programmed cell death (PCD). Such a transcriptional block could play a critical role in the neoplastic transformation of l(2)gl tissues.
The nuclear envelope (NE) is a double membrane physical barrier, which separates the nucleus from the cytoplasm. Underlying the NE are the nuclear lamins, which in combination with inner nuclear membrane proteins form the lamina. The lamina is crucial for maintaining the structural integrity of the nucleus and for positioning of nuclear pore complexes (NPCs) within the NE. The nucleoporin Nup153 has previously been reported to bind to B-type lamins. However, the specificity of this interaction is not well established. Here we show that Nup153 exhibits multiple binding sites for A- and B-type lamins. Using GST-pull down assays, we found that both the N-terminal domain of Nup153 and its C terminus associate with the Ig-fold domain of A- and B-type lamins. By employing purified Nup153 and lamin proteins in blot overlay assays we revealed that both the N-terminal and the C-terminal domain of Nup153 are directly interacting with the lamins. Moreover, we provide evidence that mutations in the lamin A Ig-fold domain selectively affect Nup153-binding, suggesting that Nup153 may play a role in lamin-associated diseases, known as laminopathies. Together our results indicate a far more intricate interplay between Nup153 and nuclear lamins than previously accepted.