Matrix attachment regions (MARs) are thought to separate chromatin into topologically constrained loop domains. A MAR located 5' of the human beta-interferon gene becomes stably base-unpaired under superhelical strain, as do the MARs flanking the immunoglobulin heavy chain gene enhancer; in both cases a nucleation site exists for DNA unwinding. Concatemerized oligonucleotides containing the unwinding nucleation site exhibited a strong affinity for the nuclear scaffold and augmented SV40 promoter activity in stable transformants. Mutated concatemerized oligonucleotides resisted unwinding, showed weak affinity for the nuclear scaffold, and did not enhance promoter activity. These results suggest that the DNA feature capable of relieving superhelical strain is important for MAR functions.

DNA topoisomerase (topo) II catalyses topological genomic changes essential for many DNA metabolic processes. It is also regarded as a structural component of the nuclear matrix in interphase and the mitotic chromosome scaffold. Mammals have two isoforms (α and β) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIα and IIβ behaved similarly in interphase but differently in mitosis, where only topo IIα was chromosome associated to a major part. During interphase, both isozymes joined in nucleolar reassembly and accumulated in nucleoli, which seemed not to involve catalytic DNA turnover because treatment with teniposide (stabilizing covalent catalytic DNA intermediates of topo II) relocated the bulk of the enzymes from the nucleoli to nucleoplasmic granules. Photobleaching revealed that the entire complement of both isozymes was completely mobile and free to exchange between nuclear subcompartments in interphase. In chromosomes, topo IIα was also completely mobile and had a uniform distribution. However, hypotonic cell lysis triggered an axial pattern. These observations suggest that topo II is not an immobile, structural component of the chromosomal scaffold or the interphase karyoskeleton, but rather a dynamic interaction partner of such structures.

For a long time S/MARs could only be characterized by the assays in vitro that led to their detection.. Only recently a number of biological activities emerged which are common to most or all S/MARs that are detected by the classical procedures. This review will focus on the phenomenon of transcriptional augmentation which is found for genomically anchored or episomal genes and on a group of partially overlapping activities which are suited to maintain an episomal status. It is further attempted to correlate properties of the S/MAR-scaffold interaction with prominent or prototype protein binding partners.Keywords: base-unpairing region (BUR), chromatin domains, episomal vectors, scaffold/matrix attached regions, stress-induced duplex destabilization (SIDD), transcriptional augmentation, unwinding elements (UE). I Introduction: Biological Activities associated with S/MARsThe proteinaceous intranuclear framework, called either `nuclear matrixA (Berezney and Coffey, 1974) or `nuclear scaffoldA (Mirkovitch et al., 1984), is thought to mediate the domain organization of the eukaryotic nucleus. Branched core filaments provide a supporting structure for the formation of DNA loops and participate in diverse matrix-supported processes such as DNA replication, -transcription and -recombination, RNA-processing and -transport as well as signal transduction and apoptotic events (review: Berezney et al., 1995).The DNA elements which mediate the attachment of chromatin loops, so called scaffold/matrix attached regions (S/MARs), have attracted considerable interest due a number of rather distinct structure-function relationships. S/MARs of several kilobasepairs are found at the borders of chromatin domains, and shorter elements with basically the same physicochemical properties occur in close association with certain enhancers or in introns. Accordingly, S/MARs are found either in nontranscribed regions or within transcription units, but rarely if ever in coding regions.A wide range of activities has been ascribed to S/MARs, among these an insulator function whereby two of these elements, bracketing a transcription unit, uncouple the gene from chromosome position effects (reviewed in Bode et al., 1998) and a function as recombination hotspots which involves nuclear matrix functions (Strissel et al., 1998). Our review will concentrate 2 on two aspects which have already received wide acceptance: the transcriptional (`augmentingA) activity of S/MARs and their apparent function(s) in episomes. In addition we will discuss some characterized protein binding partners and their possible contribution to these effects. II Characteristics of S/MAR-Scaffold RecognitionS/MARs have been operationally defined according to the protocols that lead to their detection. There are two basic criteria: first, S/MARs constitute those endogenous DNA fragments that co-purify with the nuclear matrix (i.e. remain bound to the nuclear matrix after chromatin proteins and DNA in the chromatin loops have been removed) or second, S/MARs represent th...

On its upstream side, the human interferon-beta gene is flanked by a 7-kb SAR (scaffold-attached region) DNA element. The core of this element is determined and subjected to in vitro reassociations with isolated scaffolds. Binding properties of SAR fragments with decreasing length are quantified and related to consensus sequences like the topoisomerase II box and an ATATTT motif. Characteristics as the stoichiometry, affinity, and cooperativity of the binding process are shown to depend on the length of SAR DNA and suggest a model involving a multiple-site attachment to protein scaffolds. We propose a rational approach for predicting the SAR mediated transcriptional enhancements in vivo from their binding properties in a standardized in vitro assay. The efficiency of this approach is demonstrated for a marker (huIFN-beta) and a selector gene (neor).

Topoisomerases are essential for DNA replication in dividing cells, but their genomic targets and function in postmitotic cells remain poorly understood. Here we show that a switch in the expression from Topoisomerases IIα (Top2α) to IIβ (Top2β) occurs during neuronal differentiation in vitro and in vivo. Genome-scale location analysis in stem cell-derived postmitotic neurons reveals Top2β binding to chromosomal sites that are methylated at lysine 4 of histone H3, a feature of regulatory regions. Indeed Top2β-bound sites are preferentially promoters and become targets during the transition from neuronal progenitors to neurons, at a time when cells exit the cell cycle. Absence of Top2β protein or its activity leads to changes in transcription and chromatin accessibility at many target genes. Top2β deficiency does not impair stem cell properties and early steps of neuronal differentiation but causes premature death of postmitotic neurons. This neuronal degeneration is caused by up-regulation of Ngfr p75, a gene bound and repressed by Top2β. These findings suggest a chromatin-based targeting of Top2β to regulatory regions in the genome to govern the transcriptional program associated with neuronal differentiation and longevity.epigenetic regulation | neurogenesis | gene expression | genomewide assays T opoisomerases are essential for solving topological problems arising from DNA-templated processes such as replication, transcription, recombination, chromatin remodeling, chromosome condensation, and segregation (1-5). The type I subfamily of topoisomerases achieves this task by passing one strand of the DNA through a break in the opposing strand; proteins in the type II subfamily pass a region of duplex strands from the same or a different molecule through a double-stranded gap generated in DNA (1-5). Mammalian cells encode two isozymes of type II enzymes that have highly homologous N-terminal ATPase and central core domains but differ at their C-termini (6). These two isozymes, Topoisomerases IIα (Top2α) and IIβ (Top2β), have almost identical enzymatic properties in vitro (7, 8); however, their expression patterns are dissimilar. Top2α is the main isoform expressed in proliferating cells, shows high expression in S/G2/M phases of the cell cycle, and plays important roles in DNA replication and chromosome condensation/segregation during the cell cycle (9-12).The cellular functions of Top2β are much less well understood. It is expressed in all mammalian cells throughout the cell cycle but is up-regulated robustly when cells reach a postmitotic state of terminal differentiation (13-15). For example, the postmitotic granule cells in the external germinal layer of the developing rat cerebellum show a transition from Top2α to Top2β (14), and blocking Top2β catalytic activity affects the expression of about one third of genes induced during differentiation of rat cerebellar granule neurons (16). Genetic deletion of Top2b in mice causes neural defects including aberrant axonal elongation and branching and perinatal death e...

Tyrosyl DNA phosphodiesterase 1 (TDP1) is a repair enzyme that removes adducts, e.g. of topoisomerase I from the 3-phosphate of DNA breaks. When expressed in human cells as biofluorescent chimera, TDP1 appeared more mobile than topoisomerase I, less accumulated in nucleoli, and not chromosome-bound at early mitosis. Upon exposure to camptothecin both proteins were cleared from nucleoli and rendered less mobile in the nucleoplasm. However, with TDP1 this happened much more slowly reflecting most likely the redistribution of nucleolar structures upon inhibition of rDNA transcription. Thus, a steady association of TDP1 with topoisomerase I seems unlikely, whereas its integration into repair complexes assembled subsequently to the stabilization of DNA⅐topoisomerase I intermediates is supported. Cells expressing GFP-tagged TDP1 > 100-fold in excess of endogenous TDP1 exhibited a significant reduction of DNA damage induced by the topoisomerase I poison camptothecin and could be selected by that drug. Surprisingly, DNA damage induced by the topoisomerase II poison VP-16 was also diminished to a similar extent, whereas DNA damage independent of topoisomerase I or II was not affected. Overexpression of the inactive mutant GFP-TDP1 H263A at similar levels did not reduce DNA damage by camptothecin or VP-16. These observations confirm a requirement of active TDP1 for the repair of topoisomerase I-mediated DNA damage. Our data also suggest a role of TDP1 in the repair of DNA damage mediated by topoisomerase II, which is less clear. Since overexpression of TDP1 did not compromise cell proliferation, it could be a pleiotropic resistance mechanism in cancer therapy.Tyrosyl DNA phosphodiesterase 1 (TDP1) 1 is an enzyme capable of hydrolyzing phosphodiester bonds between tyrosine and the 3Ј-phosphate of DNA (1, 2), which are typically generated in a transient manner by DNA topoisomerase I (topo I) (3). In keeping with this, yeast deletion mutations of TDP1 are deficient in the repair of DNA damage induced by camptothecin, a drug that stabilizes the transient topo I⅐DNA intermediate (2, 4 -6). More precisely, TDP1 has been characterized in these studies as a non-exclusive effector upstream of Rad52 that removes structurally modified topo I adducts (7, 8) as well as oxidative adducts (9) from the 3Ј-phosphate of a DNA break prior to homologous recombination repair. In mammals, TDP1 is (in addition or instead?) involved in an XRCC-dependent single-stranded DNA repair pathway also directed at topo I⅐DNA adducts (10 -12). Despite all the evidence of yeast deletion studies implying TDP1 in DNA repair, a familial disease caused by a mutation in the active site of the human ortholog of the enzyme exhibits a phenotype not at all typical for inadequate DNA repair, namely a slow onset of neuronal degeneration (13). This unexpected finding has prompted speculations that at least in mammals TDP1 could serve a much broader scope of functions, some of which may not even depend on catalytic activity. To further clarify the importance of TDP1 for...

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Topoisomerase II removes supercoils and catenanes generated during DNA metabolic processes such as transcription and replication. Vertebrate cells express two genetically distinct isoforms (α and β) with similar structures and biochemical activities but different biological roles. Topoisomerase IIα is essential for cell proliferation, whereas topoisomerase IIβ is required only for aspects of nerve growth and brain development. To identify the structural features responsible for these differences, we exchanged the divergent C-terminal regions (CTRs) of the two human isoforms (α 1173-1531 and β 1186-1621) and tested the resulting hybrids for complementation of a conditional topoisomerase IIα knockout in human cells. Proliferation was fully supported by all enzymes bearing the α CTR. The α CTR also promoted chromosome binding of both enzyme cores, and was by itself chromosome-bound, suggesting a role in enzyme targeting during mitosis. In contrast, enzymes bearing the β CTR supported proliferation only rarely and when expressed at unusually high levels. A similar analysis of the divergent N-terminal regions (α 1-27 and β 1-43) revealed no role in isoform-specific functions. Our results show that it is the CTRs of human topoisomerase II that determine their isoform-specific functions in proliferating cells. They also indicate persistence of some functional redundancy between the two isoforms.