A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome

The expression of genes is regulated at many levels. Perhaps the area in which least is known is how nuclear organization influences gene expression. Studies of higher-order chromatin arrangements and their dynamic interactions with other nuclear components have been boosted by recent technical advances. The emerging view is that chromosomes are compartmentalized into discrete territories. The location of a gene within a chromosome territory seems to influence its access to the machinery responsible for specific nuclear functions, such as transcription and splicing. This view is consistent with a topological model for gene regulation.

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Chromosome territories, nuclear architecture and gene regulation in mammalian cells.

tion of gene expression and other nuclear functions — namely the architecture of the nucleus as a whole. In particular, we describe evidence for a compartmentalized nuclear architecture in the mammalian cell nucleus based on chromosome territories (CTs) and an interchromatin compartment (IC) that contains macromolecular complexes that are required for replication, transcription, splicing and repair (summarized in FIG. 1). Other nuclear components, such as the nucleolus, nuclear lamina and pores, are not reviewed here (for reviews, see REFS 15,16), and although the focus of this review is the mammalian nucleus, the nuclear architecture of other organisms will be mentioned where appropriate. During the past two decades, various new methods have expanded the cell biologist’s ‘toolkit’ for the study of nuclear architecture and function (BOX 1). These methods have provided the basis for detailed studies of CTs, as well as for studies of the topology and dynamics of non-chromatin domains in the nucleus of fixed and, more recently, living cells. Computer simulations of CTs and nuclear architecture are also being used to make quantitative predictions that can be tested experimentally. On the basis of Despite all the celebrations associated with the sequencing of the human genome, and the genomes of other model organisms, our abilities to interpret genome sequences are quite limited. For example, we cannot understand the orchestrated activity — and the silencing — of many thousands of genes in any given cell just on the basis of DNA sequences, such as promoter and enhancer elements. How are the profound differences in gene activities established and maintained in a large number of cell types to ensure the development and functioning of a complex multicellular organism? To answer this question fully, we need to understand how genomes are organized in the nucleus, the basic principles of nuclear architecture and the changes in nuclear organization that occur during cellular differentiation. During recent years, EPIGENETIC mechanisms of gene regulation, such as DNA methylation and histone modification, have entered the centre stage of chromatin research. Modifications of DNA and nucleosomes, however, as well as boundaries and insulators, that affect gene regulation at the chromatin level are not the focus of this article. Instead, we review experimental data and models for a higher level of the regulaCHROMOSOME TERRITORIES, NUCLEAR ARCHITECTURE AND GENE REGULATION IN MAMMALIAN CELLS

Nuclear architecture and gene regulation in mammalian cells

We have generated and characterized a novel site-specific antibody highly specific for the phosphorylated form of the amino-terminus of histone H3 (Ser10). In this study, we used this antibody to examine in detail the relationship between H3 phosphorylation and mitotic chromosome condensation in mammalian cells. Our results extend previous biochemical studies by demonstrating that mitotic phosphorylation of H3 initiates nonrandomly in pericentromeric heterochromatin in late G2 interphase cells. Following initiation, H3 phosphorylation appears to spread throughout the condensing chromatin and is complete in most cell lines just prior to the formation of prophase chromosomes, in which a phosphorylated, but nonmitotic, chromosomal organization is observed. In general, there is a precise spatial and temporal correlation between H3 phosphorylation and initial stages of chromatin condensation. Dephosphorylation of H3 begins in anaphase and is complete immediately prior to detectable chromosome decondensation in telophase cells. We propose that the singular phosphorylation of the amino-terminus of histone H3 may be involved in facilitating two key functions during mitosis: (1) regulate protein-protein interactions to promote binding of trans-acting factors that "drive" chromatin condensation as cells enter M-phase and (2) coordinate chromatin decondensation associated with M-phase.

Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation.

Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9

A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive sequences to nontargeted chromosomes can be markedly suppressed under appropriate preannealing conditions. The remaining single-stranded DNA is hybridized to specimens of interest and detected with fluorescent or enzymelabeled avidin conjugates following post-hybridization washes. DNA inserts from recombinant libraries for chromosomes 1, 4, 7, 8, 13, 14, 18, 20, 21, 22, and X were assessed for their ability to decorate specifically their cognate chromosome; most libraries proved to be highly specific. Quantitative densitometric analyses indicated that the ratio of specific to nonspecific hybridization signal under optimal preannealing conditions was at least 8:1. Interphase nuclei showed a cohesive territorial organization of chromosomal domains, and laserscanning confocal fluorescence microscopy was used to aid the 3-D visualization of these domains. This method should be useful for both karyotypic studies and for the analysis of chromosome topography in interphase cells.

https://epub.ub.uni-muenchen.de/9279/1/cremer_thomas_9279.pdf

Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries

A classification is proposed that unifies and organizes spinal and cranial dural arteriovenous fistulous malformations (AVFMs) into three types based upon their anatomical similarities. Type I dural AVFMs drain directly into dural venous sinuses or meningeal veins. Type II malformations drain into dural sinuses or meningeal veins but also have retrograde drainage into subarachnoid veins. Type III malformations drain into subarachnoid veins and do not have dural sinus or meningeal venous drainage. The arterial supply in each of these three types is derived from meningeal arteries. The anatomical basis of the proposed classification is presented with several cases that illustrate the three types of dural AVFMs. A rationale for the treatment of spinal and cranial dural AVFMs according to their anatomical characteristics is discussed.

A proposed classification for spinal and cranial dural arteriovenous fistulous malformations and implications for treatment

Chromosome aberrations in two glioma cell lines were analyzed using biotinylated DNA library probes that specifically decorate chromosomes 1, 4, 7, 18 and 22 from pter to qter. Numerical changes, deletions and rearrangements of these chromosomes were radily visualized in metaphase spreads, as well as in early prophase and interphase nuclei. Complete chromosomes, deleted chromosomes and segments of translocated chromosomes were rapidly delineated in very complex karyotypes. Simultaneous hybridizations with additional subregional probes were used to further define aberrant chromosomes. Digital image analysis was used to quantitate the total complement of specific chromosomal DNAs in individual metaphase and interphase cells of each cell line. In spite of the fact that both glioma lines have been passaged in vitro for many years, an under-representation of chromosome 22 and an over-representation of chromosome 7 (specifically 7p) were observed. These observations agree with previous studies on gliomas. In addition, sequences of chromosome 4 were also found to be under-represented, especially in TC 593. These analyses indicate the power of these methods for pinpointing chromosome segments that are altered in specific types of tumors.

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Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes

Specific chromosome domains in interphase nuclei of neurons and glia were studied by three-dimensional (3-D) reconstruction of serial optical sections from in situ hybridized human CNS tissue. Overall patterns of centromere organization, delineated with alphoid repeats, were comparable to those seen in mouse, and are clearly conserved in mammalian evolution. Cloned probes from other individual chromosome domains were used to define interphase organization more precisely. Homologous chromosomes were spatially separated in nuclei. In large neurons, probes specific for 9q12, or 1q12 showed that at least one homolog was always compartmentalized together with centromeres on the nucleolus, while the second signal either abutted the nucleolus or was on the nuclear membrane. A telomeric Yq12 sequence also localized together with perinucleolar centromeres in a completely non-Rabl orientation. In astrocytes, these three chromosome regions were on the membrane and not necessarily associated with nucleoli. Therefore there are different patterns of interphase chromosome organization in functionally distinct cell types. In contrast to the above domains, a 1p36.3 telomeric sequence embedded in a large Alu-rich and early replicating chromosome region, was always found in an interior euchromatic nuclear compartment in both neurons and glial cells. In double hybridizations with 1q12 and 1p36.3 probes, 1p arms were clearly separated in all cells, and arms projected radially into the interior nucleoplasm with non-Rabl orientations. There was no absolute or rigid position for each 1p arm with respect to each other or to the major dendrite, indicating that individual chromosome arms may be dynamically positioned even in highly differentiated cell types. We suggest that centromeric and other highly repeated non-transcribed sequence domains may act as general organizing centers for cell type specific interphase patterns that are conserved in mammalian evolution. Such centers would allow selected groups of chromosome arms to extend into (and contract from) an interior, presumably transcriptionally active, nuclear compartment.

Reproducible compartmentalization of individual chromosome domains in human CNS cells revealed by in situ hybridization and three-dimensional reconstruction.

The position of selected chromosomes was assessed in samples of normal and epileptic human cortex with biotinylated probes specific for individual chromosome domains. Optical sectioning provided a rapid method for three-dimensional resolution of in situ hybridization signals in interphase cells, and solid models were reconstructed from digitized images for detailed rotational studies. There was a dramatic repositioning of the X chromosome in neurons of both males and females in electrophysiologically defined seizure foci. Other chromosomes (1, 9, and Y) showed more subtle positional changes. Specifically altered nuclear patterns involving the X chromosome may become established and create the genetic memory for intractable seizure activity.

https://science.sciencemag.org/content/sci/242/4886/1687.full.pdf

Jonathan A. Borden

Papers

Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries

A proposed classification for spinal and cranial dural arteriovenous fistulous malformations and implications for treatment

Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes

Reproducible compartmentalization of individual chromosome domains in human CNS cells revealed by in situ hybridization and three-dimensional reconstruction.

Movement of the X chromosome in epilepsy