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ABSTRACT: There is rapidly growing evidence that folding of the chromatin fibre inside the interphase nucleus has an important role in the regulation of gene expression. In particular, the formation of loops mediated by the interaction between specific regulatory elements, for instance enhancers and promoters, is crucial in gene control. Biochemical studies that were based on the chromosome conformation capture (3C) technology have confirmed that eukaryotic genomes are highly looped. Insight into the underlying principles comes from polymer models that explore the properties of the chromatin fibre inside the nucleus. Recent models indicate that chromatin looping can explain various properties of interphase chromatin, including chromatin compaction and compartmentalisation of chromosomes. Entropic effects have a key role in these models. In this Commentary, we give an overview of the recent conjunction of ideas regarding chromatin looping in the fields of biology and polymer physics. Starting from simple linear polymer models, we explain how specific folding properties emerge upon introducing loops and how this explains a variety of experimental observations. We also discuss different polymer models that describe chromatin folding and compare them to experimental data. Experimentally testing the predictions of such polymer models and their subsequent improvement on the basis of measurements provides a solid framework to begin to understand how our genome is folded and how folding relates to function.
Journal of Cell Science 03/2011; 124(Pt 6):839-45. · 6.11 Impact Factor
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ABSTRACT: The precise localization of transcribed DNA and resulting RNA is an important aspect of the functional architecture of the nucleus. To this end we have developed a novel in situ hybridization approach in combination with immunoelectron microscopy, using sense and anti-sense RNA probes that are derived from total cellular or cytoplasmic poly(A+) RNA. This new technology is much more gentle than classical in situ hybridization using DNA probes and shows excellent preservation of nuclear structure. Carried out on ultrathin sections of fixed and resin-embedded COS-7 cells, it revealed at high resolution the localization of the genes that code for the cellular mRNAs. Quantitative analysis shows that most transcribed DNA is concentrated in the perichromatin region, i.e. the interface between subchromosomal compact chromatin domains and the interchromatin space essentially devoid of DNA. The RNA that is produced is found mainly in the perichromatin region and the interchromatin space. These results imply that in the mammalian nucleus the chromatin fiber is folded so that active genes are predominantly present in the perichromatin region, which is the most prominent site of transcription.
Experimental Cell Research 11/2010; 317(4):433-44. · 3.58 Impact Factor
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ABSTRACT: Episomal vectors assembled from defined genetic components are a promising alternative to traditional gene therapy vectors that integrate in the host genome and may cause insertional mutations. The vector pEPI-eGFP is stably retained in the episomal state in cultured mammalian cells at low copy number for many generations without integration into the host genome. Although pEPI-eGFP is a fully engineered vector, little is known about how it interacts with the host genome and about the molecular mechanisms that are responsible for its transcriptional activity. We have analyzed the expression of the episomal reporter gene eGFP under conditions that affect the chromatin state of the genome. We have also constructed pEPI derivatives carrying a tandem array of lac operator sequences, which allows in vivo visualization and manipulation of the chromatin state of the episome. We show that changes in chromatin state of both the host and pEPI-eGFP induces changes in episomal gene activity and influences the episome's nuclear distributions. We conclude that episomal genes are subject to control systems of the host, similarly to their counterparts in the host genome.
Chromosome Research 11/2010; 18(7):757-75. · 3.09 Impact Factor
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ABSTRACT: Development and acclimation processes to the environment are associated with large-scale changes in chromatin compaction in Arabidopsis (Arabidopsis thaliana). Here, we studied the effects of light signals on chromatin organization. A decrease in light intensity induces a large-scale reduction in chromatin compaction. This low light response is reversible and shows strong natural genetic variation. Moreover, the degree of chromatin compaction is affected by light quality signals relevant for natural canopy shade. The photoreceptor CRYPTOCHROME2 appears a general positive regulator of low light-induced chromatin decompaction. Phytochrome B also controls light-induced chromatin organization, but its effect appears to be dependent on the genetic background. We present a model in which chromatin compaction is regulated by the light environment via CRYPTOCHROME2 protein abundance, which is controlled by phytochrome B action.
Plant physiology 10/2010; 154(4):1686-96. · 6.53 Impact Factor
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ABSTRACT: Sphingomyelin is one of the major phospholipids in the cell nucleus. However, its intranuclear distribution with regard to different functional nuclear domains as well as its possible involvement in the nuclear functional architecture remains to be elucidated.
We carried out an ultrastructural cytochemical study of the intranuclear distribution of SM (sphingomyelin) using an in situ binding assay of neutral SMase (sphingomyelinase) conjugated to colloidal gold particles. The enzymatic labelling was carried out on ultrathin sections of different mammalian cells prepared by means of various fixation and resin-embedding protocols. Transmission electron microscopic analysis revealed preferential localization of SM within the PR (perichromatin region), a functionally important nucleoplasmic domain containing sites of pre-mRNA synthesis and processing. In the nucleolus, SM is mostly associated with the dense fibrillar component containing transcriptionally active ribosomal genes. Microinjection of enzymatically active SMase into living cells resulted in a rapid degradation of intranuclear structure.
Our observations, supported by biochemical data, provide evidence for the involvement of SM in important nuclear functions. They bring additional information pointing out the PR as an essential functional nuclear domain. Furthermore, they suggest a role for SM in the internal nuclear architecture.
Biology of the Cell 06/2010; 102(6):361-75. · 3.60 Impact Factor
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ABSTRACT: To understand how multiprotein complexes assemble and function on chromatin, we combined quantitative analysis of the mammalian nucleotide excision DNA repair (NER) machinery in living cells with computational modeling. We found that individual NER components exchange within tens of seconds between the bound state in repair complexes and the diffusive state in the nucleoplasm, whereas their net accumulation at repair sites evolves over several hours. Based on these in vivo data, we developed a predictive kinetic model for the assembly and function of repair complexes. DNA repair is orchestrated by the interplay of reversible protein-binding events and progressive enzymatic modifications of the chromatin substrate. We demonstrate that faithful recognition of DNA lesions is time consuming, whereas subsequently, repair complexes form rapidly through random and reversible assembly of NER proteins. Our kinetic analysis of the NER system reveals a fundamental conflict between specificity and efficiency of chromatin-associated protein machineries and shows how a trade off is negotiated through reversibility of protein binding.
The Journal of Cell Biology 05/2010; 189(3):445-63. · 10.26 Impact Factor
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ABSTRACT: Summary Paramutation is the transfer of epigenetic information between alleles that leads to a heritable change in expression of one of these alleles. Paramutation at the tissue-specifically expressed maize (Zea mays) b1 locus involves the low-expressing B' and high-expressing B-I allele. Combined in the same nucleus, B' heritably changes B-I into B'. A hepta-repeat located 100-kb upstream of the b1 coding region is required for paramutation and for high b1 expression. The role of epigenetic modifications in paramutation is currently not well understood. In this study, we show that the B' hepta-repeat is DNA-hypermethylated in all tissues analyzed. Importantly, combining B' and B-I in one nucleus results in de novo methylation of the B-I repeats early in plant development. These findings indicate a role for hepta-repeat DNA methylation in the establishment and maintenance of the silenced B' state. In contrast, nucleosome occupancy, H3 acetylation, and H3K9 and H3K27 methylation are mainly involved in tissue-specific regulation of the hepta-repeat. Nucleosome depletion and H3 acetylation are tissue-specifically regulated at the B-I hepta-repeat and associated with enhancement of b1 expression. H3K9 and H3K27 methylation are tissue-specifically localized at the B' hepta-repeat and reinforce the silenced B' chromatin state. The B' coding region is H3K27 dimethylated in all tissues analyzed, indicating a role in the maintenance of the silenced B' state. Taken together, these findings provide insight into the mechanisms underlying paramutation and tissue-specific regulation of b1 at the level of chromatin structure.
The Plant Journal 04/2010; · 6.16 Impact Factor
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Federico Tessadori,
Martijn van Zanten,
Penka Pavlova,
Rachel Clifton,
Frédéric Pontvianne,
L Basten Snoek,
Frank F Millenaar,
Roeland Kees Schulkes, Roel van Driel,
Laurentius A C J Voesenek,
Charles Spillane,
Craig S Pikaard,
Paul Fransz,
Anton J M Peeters
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ABSTRACT: Natural genetic variation in Arabidopsis thaliana exists for many traits and often reflects acclimation to local environments. Studying natural variation has proven valuable in the characterization of phenotypic traits and, in particular, in identifying genetic factors controlling these traits. It has been previously shown that chromatin compaction changes during development and biotic stress. To gain more insight into the genetic control of chromatin compaction, we investigated the nuclear phenotype of 21 selected Arabidopsis accessions from different geographic origins and habitats. We show natural variation in chromatin compaction and demonstrate a positive correlation with latitude of geographic origin. The level of compaction appeared to be dependent on light intensity. A novel approach, combining Quantitative Trait Locus (QTL) mapping and microscopic examination, pointed at PHYTOCHROME-B (PHYB) and HISTONE DEACETYLASE-6 (HDA6) as positive regulators of light-controlled chromatin compaction. Indeed, mutant analyses demonstrate that both factors affect global chromatin organization. HDA6, in addition, strongly promotes the light-mediated compaction of the Nucleolar Organizing Regions (NORs). The accession Cape Verde Islands-0 (Cvi-0), which shows sequence polymorphism in the PHYB gene and in the HDA6 promotor, resembles the hda6 mutant in having reduced chromatin compaction and decreased methylation levels of DNA and histone H3K9 at the NORs. We provide evidence that chromatin organization is controlled by light intensity. We propose that chromatin plasticity is associated with acclimation of Arabidopsis to its environment. The polymorphic alleles such as PHYB and HDA6 control this process.
PLoS Genetics 10/2009; 5(9):e1000638. · 8.69 Impact Factor
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Martijn S Luijsterburg,
Christoffel Dinant,
Hannes Lans,
Jan Stap,
Elzbieta Wiernasz,
Saskia Lagerwerf,
Daniël O Warmerdam,
Michael Lindh,
Maartje C Brink,
Jurek W Dobrucki,
Jacob A Aten,
Maria I Fousteri,
Gert Jansen,
Nico P Dantuma,
Wim Vermeulen,
Leon H F Mullenders,
Adriaan B Houtsmuller,
Pernette J Verschure, Roel van Driel
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ABSTRACT: Heterochromatin protein 1 (HP1) family members are chromatin-associated proteins involved in transcription, replication, and chromatin organization. We show that HP1 isoforms HP1-alpha, HP1-beta, and HP1-gamma are recruited to ultraviolet (UV)-induced DNA damage and double-strand breaks (DSBs) in human cells. This response to DNA damage requires the chromo shadow domain of HP1 and is independent of H3K9 trimethylation and proteins that detect UV damage and DSBs. Loss of HP1 results in high sensitivity to UV light and ionizing radiation in the nematode Caenorhabditis elegans, indicating that HP1 proteins are essential components of DNA damage response (DDR) systems. Analysis of single and double HP1 mutants in nematodes suggests that HP1 homologues have both unique and overlapping functions in the DDR. Our results show that HP1 proteins are important for DNA repair and may function to reorganize chromatin in response to damage.
The Journal of Cell Biology 06/2009; 185(4):577-86. · 10.26 Impact Factor
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ABSTRACT: Live-cell imaging studies aided by mathematical modeling have provided unprecedented insight into assembly mechanisms of multiprotein complexes that control genome function. Such studies have unveiled emerging properties of chromatin-associated systems involved in DNA repair and transcription.
The Journal of Cell Biology 05/2009; 185(1):21-6. · 10.26 Impact Factor
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ABSTRACT: This work examines the involvement of chromatin looping in the transcriptional regulation of two epialleles of the maize (Zea mays) b1 gene, B-I and B'. These two epialleles are tissue-specifically regulated and are involved in paramutation. B-I and B' are expressed at high and low levels, respectively. A hepta-repeat approximately 100 kb upstream of the transcription start site (TSS) is required for both paramutation and high b1 expression. Using chromosome conformation capture, we show that the hepta-repeat physically interacts with the TSS region in a tissue- and expression level-specific manner. Multiple repeats are required to stabilize this interaction. High b1 expression is mediated by a multiloop structure; besides the hepta-repeat, other sequence regions physically interact with the TSS as well, and these interactions are epiallele- and expression level-specific. Formaldehyde-assisted isolation of regulatory elements uncovered multiple interacting regions as potentially regulatory.
The Plant Cell 04/2009; 21(3):832-42. · 8.99 Impact Factor
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Julio Mateos-Langerak,
Manfred Bohn,
Wim de Leeuw,
Osdilly Giromus,
Erik M M Manders,
Pernette J Verschure,
Mireille H G Indemans,
Hinco J Gierman,
Dieter W Heermann, Roel van Driel,
Sandra Goetze
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ABSTRACT: Genome function in higher eukaryotes involves major changes in the spatial organization of the chromatin fiber. Nevertheless, our understanding of chromatin folding is remarkably limited. Polymer models have been used to describe chromatin folding. However, none of the proposed models gives a satisfactory explanation of experimental data. In particularly, they ignore that each chromosome occupies a confined space, i.e., the chromosome territory. Here, we present a polymer model that is able to describe key properties of chromatin over length scales ranging from 0.5 to 75 Mb. This random loop (RL) model assumes a self-avoiding random walk folding of the polymer backbone and defines a probability P for 2 monomers to interact, creating loops of a broad size range. Model predictions are compared with systematic measurements of chromatin folding of the q-arms of chromosomes 1 and 11. The RL model can explain our observed data and suggests that on the tens-of-megabases length scale P is small, i.e., 10-30 loops per 100 Mb. This is sufficient to enforce folding inside the confined space of a chromosome territory. On the 0.5- to 3-Mb length scale chromatin compaction differs in different subchromosomal domains. This aspect of chromatin structure is incorporated in the RL model by introducing heterogeneity along the fiber contour length due to different local looping probabilities. The RL model creates a quantitative and predictive framework for the identification of nuclear components that are responsible for chromatin-chromatin interactions and determine the 3-dimensional organization of the chromatin fiber.
Proceedings of the National Academy of Sciences 03/2009; 106(10):3812-7. · 9.68 Impact Factor
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ABSTRACT: Gene regulation in higher eukaryotes frequently involves physical interactions between genomic sequence elements tens of kilobases apart on the same chromosome but can also entail interactions between different chromosomes. Chromosome Conformation Capture (3C) is a powerful tool to identify such interactions. 3C technology is based on formaldehyde crosslinking of chromatin, followed by restriction digestion and intramolecular ligation. Quantitative detection of ligation products by PCR (qPCR; not discussed in this protocol) provides insight into the interaction frequencies between chromosomal fragments and thereby the spatial organization of a genomic region. Detailed 3C protocols have been published for yeast and mammals. However, these protocols cannot simply be transferred to plant tissues. In this paper, we provide a maize-specific 3C protocol and present a general strategy to systematically optimize the protocol for other plants. Once the technique and appropriate controls are established, the 3C procedure (including qPCR) can be completed in 5-7 d.
Nature Protocol 02/2009; 4(8):1216-29. · 8.36 Impact Factor
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ABSTRACT: Nucleotide excision repair (NER) is an evolutionary conserved DNA repair system that is essential for the removal of UV-induced DNA damage. In this study we investigated how NER is compartmentalized in the interphase nucleus of human cells at the ultrastructural level by using electron microscopy in combination with immunogold labeling. We analyzed the role of two nuclear compartments: condensed chromatin domains and the perichromatin region. The latter contains transcriptionally active and partly decondensed chromatin at the surface of condensed chromatin domains. We studied the distribution of the damage-recognition protein XPC and of XPA, which is a central component of the chromatin-associated NER complex. Both XPC and XPA rapidly accumulate in the perichromatin region after UV irradiation, whereas only XPC is also moderately enriched in condensed chromatin domains. These observations suggest that DNA damage is detected by XPC throughout condensed chromatin domains, whereas DNA-repair complexes seem preferentially assembled in the perichromatin region. We propose that UV-damaged DNA inside condensed chromatin domains is relocated to the perichromatin region, similar to what has been shown for DNA replication. In support of this, we provide evidence that UV-damaged chromatin domains undergo expansion, which might facilitate the translocation process. Our results offer novel insight into the dynamic spatial organization of DNA repair in the human cell nucleus.
Journal of Cell Science 01/2009; 122(Pt 1):83-91. · 6.11 Impact Factor
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ABSTRACT: The genomic DNA of all organisms across the three kingdoms of life needs to be compacted and functionally organized. Key players in these processes are DNA supercoiling, macromolecular crowding and architectural proteins that shape DNA by binding to it. The architectural proteins in bacteria, archaea and eukaryotes generally do not exhibit sequence or structural conservation especially across kingdoms. Instead, we propose that they are functionally conserved. Most of these proteins can be classified according to their architectural mode of action: bending, wrapping or bridging DNA. In order for DNA transactions to occur within a compact chromatin context, genome organization cannot be static. Indeed chromosomes are subject to a whole range of remodeling mechanisms. In this review, we discuss the role of (i) DNA supercoiling, (ii) macromolecular crowding and (iii) architectural proteins in genome organization, as well as (iv) mechanisms used to remodel chromosome structure and to modulate genomic activity. We conclude that the underlying mechanisms that shape and remodel genomes are remarkably similar among bacteria, archaea and eukaryotes.
Critical Reviews in Biochemistry and Molecular Biology 12/2008; 43(6):393-418. · 7.66 Impact Factor
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ABSTRACT: Analysis of the genome organization of higher eukaryotes indicates that it contains many clusters of functionally related genes. In these clusters, the activity of a single gene is regulated hierarchically at a local gene-level and a global cluster-level. Whether a single gene can be activated by a dedicated transcription factor depends on the epigenetic status of the cluster, i.e. whether it is epigenetically permissive or silenced. The consequence of gene clusters for the functioning of gene networks is largely unexplored. The accumulating biological knowledge about mechanisms for epigenetic regulation, signal transduction, and gene clusters makes such explorations a timely challenge. We explore the steady-state behavior of two gene clusters that mutually inhibit each other. This gives rise to multiple steady states in this simple system of interacting clusters. We illustrate that a gene cluster encoding a module composed of a signal transduction network and a transcription factor can generate versatile temporal dynamics that resembles cellular differentiation. The gene cluster can be epigenetically silenced and activated by a dedicated transcription factor. This module displays transient signal sensitivity, and irreversible decisions (commitment; hysteresis) depending on the identity and temporal sequence of external signals.
Journal of Theoretical Biology 07/2008; 252(3):482-7. · 2.21 Impact Factor
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ABSTRACT: Gene regulation in higher eukaryotes has been shown to involve regulatory sites, such as promoters and enhancers which act at the level of individual genes, and mechanisms which control the functional state of gene clusters. A fundamental question is whether additional levels of genome control exist. Nuclear organization and large-scale chromatin structure may constitute such a level and play an important role in the cell-type specific orchestration of the expression of thousands of genes in eukaryotic cells. Numerous observations indicate a tight correlation between genome activity and nuclear and large-scale chromatin structure. However, causal relationships are rare. Here we explore how these might be uncovered.
Journal of Cellular Biochemistry 01/2008; 102(5):1067-75. · 2.87 Impact Factor
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ABSTRACT: Remarkably little is known about the higher-order folding motifs of the chromatin fiber inside the cell nucleus. Folding depends among others on local gene density and transcriptional activity and plays an important role in gene regulation. Strikingly, at fiber lengths above 5 to 10 Mb the measured mean square distance between any two points on the chromatin fiber is independent of polymer length. We propose a polymer model that can explain this leveling-off by means of random looping. We derive an analytical expression for the mean square displacement between two arbitrary beads. Here the average is taken over the thermal ensemble with a fixed but random loop configuration, while quenched averaging over the ensemble of different loop configurations--which turns out to be equivalent to averaging over an ensemble of random matrices--is performed numerically. A detailed investigation of this model shows that loops on all scales are necessary to fit experimental data.
Physical Review E 11/2007; 76(5 Pt 1):051805. · 2.26 Impact Factor
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ABSTRACT: The perichromatin region has emerged as an important functional domain of the interphase nucleus. Major nuclear functions, such as DNA replication and transcription, as well as different RNA processing factors, occur within this domain. In this review, we summarize in situ observations regarding chromatin structure analysed by transmission electron microscopy and compare results to data obtained by other methods. In particular, we address the functional architecture of the perichromatin region and the way chromatin may be folded within this nucleoplasmic domain.
Seminars in Cell and Developmental Biology 11/2007; 18(5):676-81. · 6.65 Impact Factor
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ABSTRACT: Higher order chromatin structure, i.e. the three-dimensional (3D) organization of the genome in the interphase nucleus, is an important component in the orchestration of gene expression in the mammalian genome. In this review we describe principles of higher order chromatin structure discussing three organizational parameters, i.e. chromatin folding, chromatin compaction and the nuclear position of the chromatin fibre. We argue that principles of 3D genome organization are probabilistic traits, reflected in a considerable cell-to-cell variation in 3D genome structure. It will be essential to understand how such higher order organizational aspects contribute to genome function to unveil global genome regulation.
Seminars in Cell and Developmental Biology 11/2007; 18(5):707-14. · 6.65 Impact Factor
