Biomedical subjects

Pernette J Verschure

Publications and source records attributed to Pernette J Verschure.

12 recordsLinked to original sources

PubMed2006

Chromosome organization and gene control: it is difficult to see the picture when you are inside the frame.

The organization of the genome in the nucleus is related to its function. The functional compartmentalization of the genome is described at the nuclear, chromosomal, subchromosomal, nucleosomal, and DNA sequence level. These descriptions originate from the techniques that were used for analysis. The different levels of compartmentalization are not easily reconciled, because the techniques applied to identify genome compartmentalization generally cannot be performed in combination. We have obtained a large body of information on individual "actors" and "scenes" in the nucleus regarding genome compartmentalization, but we still do not understand how and by what pieces of equipment the "actors" play their game. The next challenge is to understand the combined operation of the various levels of functional genome organization in the nucleus, that is, how do the epigenetic and genetic levels act together. In this paper, I will highlight some of the general features and observations of functional organization of the eukaryotic genome in interphase nuclei and discuss the concepts and views based on observed correlations between genome organization and function. I will reflect on what is to be expected from this field of research when the functional levels of genome compartmentalization are integrated. In this context I will draw attention to what might be needed to improve our understanding.

Cell Nucleus↗

PubMed2006

Step into the groove: engineered transcription factors as modulators of gene expression.

Increasing knowledge about the influence of dysregulated gene expression in causing numerous diseases opens up new possibilities for the development of innovative therapeutics. In this chapter, we first describe different mechanisms of misregulated gene expression resulting in various pathophysiological conditions. Then, an overview is given of different technologies developed to readjust expression levels of genes. One of the most promising upcoming approaches in this respect is the development of engineered zinc-finger transcription factors. Results obtained from modulating endogenous gene expression using such engineered transcription factors are reviewed in depth. Finally, we address possible pitfalls of using such transcriptional targeting approaches at the "chromatin level." We describe aspects of studies at this level that influence successful DNA binding of engineered transcription factors, thereby affecting gene activity. Engineered transcription factors have great promise as potent therapeutics. Moreover, this technology is expected to yield fundamental knowledge about the organization and function of the genome.

Animals↗

PubMed2006

Step out of the groove: epigenetic gene control systems and engineered transcription factors.

At the linear DNA level, gene activity is believed to be driven by binding of transcription factors, which subsequently recruit the RNA polymerase to the gene promoter region. However, it has become clear that transcriptional activation involves large complexes of many different proteins, which not only directly recruit components of the transcription machinery but also affect the DNA folding. Such proteins, including various chromatin-modifying enzymes, alter among other processes nucleosome positioning and histone modifications and are potentially involved in changing the overall structure of the chromatin and/or the position of chromatin in the nucleus. These epigenetic regulatory features are now known to control and regulate gene expression, although the molecular mechanisms still need to be clarified in more detail. Several diseases are characterized by aberrant gene-expression patterns. Many of these diseases are linked to dysregulation of epigenetic gene-regulatory systems. To interfere with aberrant gene expression, a novel approach is emerging as a disease therapy, involving engineered transcription factors. Engineered transcription factors are based on, for example, zinc-finger proteins (ZFP) that bind DNA in a sequence-specific manner. Engineered transcription factors based on ZFP are fused to effector domains that function to normalize disrupted gene-expression levels. Zinc-finger proteins most likely also influence epigenetic regulatory systems, such as the complex set of chemical histone and DNA modifications, which control chromatin compaction and nuclear organization. In this chapter, we review how epigenetic regulation systems acting at various levels of packaging the genome in the cell nucleus add to gene-expression control at the DNA level. Since an increasing number of diseases are described to have a clear link to epigenetic dysregulation, we here highlight 10 examples of such diseases. In the second part, we describe the different effector domains that have been fused to ZFPs and are capable of activating or silencing endogenous genes, and we illustrate how these effector domains influence epigenetic control mechanisms. Finally, we speculate how accumulating knowledge about epigenetics can be exploited to make such zinc-finger-transcription factors (ZF-TF) even more effective.

Animals↗

PubMed2005

Truncated HP1 lacking a functional chromodomain induces heterochromatinization upon in vivo targeting.

Packaging of the eukaryotic genome into higher order chromatin structures is tightly related to gene expression. Pericentromeric heterochromatin is typified by accumulations of heterochromatin protein 1 (HP1), methylation of histone H3 at lysine 9 (MeH3K9) and global histone deacetylation. HP1 interacts with chromatin by binding to MeH3K9 through the chromodomain (CD). HP1 dimerizes with itself and binds a variety of proteins through its chromoshadow domain. We have analyzed at the single cell level whether HP1 lacking its functional CD is able to induce heterochromatinization in vivo. We used a lac-operator array-based system in mammalian cells to target EGFP-lac repressor tagged truncated HP1alpha and HP1beta to a lac operator containing gene-amplified chromosome region in living cells. After targeting truncated HP1alpha or HP1beta we observe enhanced tri-MeH3K9 and recruitment of endogenous HP1alpha and HP1beta to the chromosome region. We show that CD-less HP1alpha can induce chromatin condensation, whereas the effect of truncated HP1beta is less pronounced. Our results demonstrate that after lac repressor-mediated targeting, HP1alpha and HP1beta without a functional CD are able to induce heterochromatinization.

Animals↗

PubMed2005

Imaging in situ protein-DNA interactions in the cell nucleus using FRET-FLIM.

Although the distribution of DNA-binding proteins inside the cell nucleus can be analyzed by immunolabeling or by tagging proteins with GFP, we cannot establish whether the protein is bound to DNA or not. Here, we describe a novel approach that allows imaging of the in situ interaction between a GFP-fusion protein and DNA in the cell nucleus, using fluorescence resonance energy transfer (FRET). We used fluorescence lifetime imaging microscopy (FLIM) as a reliable tool to detect protein in contact with DNA. The method was successfully applied to the DNA-binding proteins histone H2B and the glucocorticoid receptor and to the heterochromatin-associated proteins HP1alpha and HP1beta.

Cell Line, Tumor↗

PubMed2005

In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation.

Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) alpha (HP1alpha) and HP1beta on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1alpha and HP1beta in vivo targeting. HP1alpha targeting causes the recruitment of endogenous HP1beta to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1alpha and HP1beta targeting is sufficient to induce heterochromatin formation.

Cells, Cultured↗

PubMed2004

Positioning the genome within the nucleus.

Higher eukaryotic genomes contain both housekeeping genes and genes of which the expression is restricted to a defined time and space. It is well established that a correlation exists between structural organization of the genome and gene expression control. The functional mechanisms underlying this correlation are still poorly understood. Here I describe several observations that are the basis of present concepts of genome organization and nuclear architecture related to functionality. Regarding the relationship between positioning and disturbed cell functionality, I describe observations showing that the proximity of selected gene loci is statistically correlated with their propensity for oncogenic translocations as well as observations of patterns occurring in neurodegenerative disorders where unstable repeats are translated into an expanded polyglutamine tract. Such observations underscore the importance to understand how genetic perturbations lead to the global reorganization of nuclear architecture, chromatin structure and widespread changes in gene expression.

Animals↗

PubMed2003

The eukaryotic genome: a system regulated at different hierarchical levels.

Eukaryotic gene expression can be viewed within a conceptual framework in which regulatory mechanisms are integrated at three hierarchical levels. The first is the sequence level, i.e. the linear organization of transcription units and regulatory sequences. Here, developmentally co-regulated genes seem to be organized in clusters in the genome, which constitute individual functional units. The second is the chromatin level, which allows switching between different functional states. Switching between a state that suppresses transcription and one that is permissive for gene activity probably occurs at the level of the gene cluster, involving changes in chromatin structure that are controlled by the interplay between histone modification, DNA methylation, and a variety of repressive and activating mechanisms. This regulatory level is combined with control mechanisms that switch individual genes in the cluster on and off, depending on the properties of the promoter. The third level is the nuclear level, which includes the dynamic 3D spatial organization of the genome inside the cell nucleus. The nucleus is structurally and functionally compartmentalized and epigenetic regulation of gene expression may involve repositioning of loci in the nucleus through changes in large-scale chromatin structure.

Animals↗

PubMed2003

Condensed chromatin domains in the mammalian nucleus are accessible to large macromolecules.

Most chromatin in interphase nuclei is part of condensed chromatin domains. Previous work has indicated that transcription takes place primarily at the surface of chromatin domains, that is, in the perichromatin region. It is possible that genes inside chromatin domains are silenced due to inaccessibility to macromolecular components of the transcription machinery. We have tested the accessibility of chromatin domains in nuclei of living cells with proteins and dextrans of different molecular sizes. Our results show that chromatin domains are readily accessible to large macromolecules, including proteins with a molecular weight of several hundred kilodaltons. Therefore, the silencing of genes that are incorporated into such domains is not due to the physical inaccessibility of condensed chromatin domains to transcription factors.

Cell Nucleus↗

PubMed2003

Polycomb group gene silencing proteins are concentrated in the perichromatin compartment of the mammalian nucleus.

Human Polycomb group (PcG) proteins are involved in cell-type-dependent epigenetic gene silencing in an evolutionarily conserved manner. We have analysed the subnuclear localisation of these regulatory proteins in two different human cell lines and in rat liver tissue by means of light and electron immunomicroscopy using specific antibodies. We find that the PcG proteins HPC2, HPH1, BMI1 and RING1 are highly concentrated in the perichromatin compartment, situated at the surface of condensed chromatin domains. This compartment was demonstrated earlier to be the nuclear site where most pre-mRNA synthesis takes place. Interestingly, these PcG proteins are virtually absent from the interior of condensed chromatin areas. The present observations therefore show that transcriptionally active and PcG-silenced loci occur within the same spatially limited nuclear domain. Our novel high-resolution data strongly support the idea that epigenetic PcG-mediated gene silencing is a local event, rather than affecting large chromatin domains. In addition to being associated with the perichromatin region, PcG proteins also occur in the interchromatin space. Implications of these observations for higher order chromatin structure and for the mechanisms of PcG-mediated gene silencing are discussed.

Animals↗

PubMed2002

Large-scale chromatin organization and the localization of proteins involved in gene expression in human cells.

Compartmentalization of the interphase nucleus is an important element in the regulation of gene expression. Here we investigated the functional organization of the interphase nucleus of HeLa cells and primary human fibroblasts. The spatial distribution of proteins involved in transcription (TFIIH and RNA polymerase II) and RNA processing and packaging (hnRNP-U) were analyzed in relation to chromosome territories and large-scale chromatin organization. We present evidence that these proteins are present predominantly in the interchromatin space, inside and between chromosome territories, and are largely excluded by domains of condensed chromatin. We show that they are present throughout the active and inactive X-chromosome territories in primary female fibroblasts, indicating that these proteins can freely diffuse throughout the interchromatin compartment in the interphase nucleus. Furthermore, we established that the in vivo spatial distribution of condensed chromatin in the interphase nucleus does not depend on ongoing transcription. Our data support a conceptually simple model for the functional organization of interphase nuclei.

Cell Compartmentation↗

PubMedDate not supplied

Isualization and analysis of large data collections: a case study applied to confocal microscopy data.

In this paper we propose an approach in which interactive visualization and analysis are combined with batch tools for the processing of large data collections. Large and heterogeneous data collections are difficult to analyze and pose specific problems to interactive visualization. Application of the traditional interactive processing and visualization approaches as well as batch processing encounter considerable drawbacks for such large and heterogeneous data collections due to the amount and type of data. Computing resources are not sufficient for interactive exploration of the data and automated analysis has the disadvantage that the user has only limited control and feedback on the analysis process. In our approach, an analysis procedure with features and attributes of interest for the analysis is defined interactively. This procedure is used for off-line processing of large collections of data sets. The results of the batch process along with "visual summaries" are used for further analysis. Visualization is not only used for the presentation of the result, but also as a tool to monitor the validity and quality of the operations performed during the batch process. Operations such as feature extraction and attribute calculation of the collected data sets are validated by visual inspection. This approach is illustrated by an extensive case study, in which a collection of confocal microscopy data sets is analyzed.

Algorithms↗