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.

Using the FLP/FRT system for site-specific recombination and the wild-type recognition site (FRT) in conjunction with certain mutant FRT sites, it becomes possible to provoke, with high yield, a double-reciprocal crossover event in cultured mammalian cells. It is demonstrated that this technology enables a targeting of expression cassettes to appropriate chromosomal reference sites in the recipient cell to improve the concepts of reverse genetics. The design of mutant FRT sites promoting such a process will be delineated. Our results show that the five spacer mutations tested are functional as the wild type but differ in the extent of their cross-recombination, which has to be minimized for their simultaneous usage.

We have developed an episomal replicating expression vector in which the SV40 gene coding for the large T-antigen was replaced by chromosomal scaffold/matrix attached regions. Southern analysis as well as vector rescue experiments in CHO cells and in Escherichia coli demonstrate that the vector replicates episomally in CHO cells. It occurs in a very low copy number in the cells and is stably maintained over more than 100 generations without selection pressure.

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).

The activation of mammalian origins of replication depends so far on ill understood epigenetic events, such as binding of transcription factors, chromatin structure, and nuclear localization. Understanding these mechanisms is not only a scientific challenge but also represents a prerequisite for the rational design of nonviral episomal vectors for mammalian cells. In this paper, we demonstrate that a tetramer of a 155-bp minimal nuclear scaffold͞matrix attached region DNA module linked to an upstream transcription unit is sufficient for replication and mitotic stability of a mammalian episome in the absence of selection. Fluorescence in situ hybridization analyses, crosslinking with cis-diammineplatinum(II)-dichloride and chromatin immunoprecipitation demonstrate that this vector associates with the nuclear matrix or scaffold in vivo by means of specific interaction of the nuclear scaffold͞matrix attached region with the nuclear matrix protein SAF-A. Results presented in this paper define the minimal requirements of an episomal vector for mammalian cells on the molecular level.DNA replication ͉ mitotic stability ͉ nuclear matrix ͉ SAF-A

The biological significance of nuclear scaffold/matrix-attachment regions (S/MARs) remains a topic of long-standing interest. The key to understanding S/MAR behavior relies on determining the physical attributes of in vivo S/MARs and whether they serve as rigid or flexible chromatin loop anchors. To analyze S/MAR behavior, single and multiple copies of the S/MAR-containing constructs were introduced into various host genomes of transgenic mice and transfected cell lines. These in vivo integration events provided a system to study the association and integration patterns of each introduced S/MAR. By utilizing FISH to visualize directly the localization of S/MARs on the nuclear matrix or chromatin loop, we were able to assign specific attributes to the S/MAR. Surprisingly, when multiple-copy S/MARs were introduced they were selected and used as nuclear matrix anchors in a discriminatory manner, even though they all contained identical primary sequences. This selection process was probably mediated by S/MAR availability including binding strength and copy number, as reflected by the expression profiles and association of multi-copy tandem inserted constructs. Whereas S/MARs functioned as the mediators of loop attachment, they were used in a selective and dynamic fashion. Consequently, S/MAR anchors were necessary but not sufficient for chromatin loops to form. These observations reconcile many seemingly contradictory attributes previously associated with S/MARs.

We have transfected DNA corresponding to the complete chromatin domain of human interferon beta (huIFN-beta) gene into mouse L cells. In this construct, which is flanked by scaffold-attached regions (SARs), the gene's transcription was enhanced 20-30-fold with respect to DNAs containing only the immediate regulatory elements. To elucidate the role of SAR elements in the transcriptional enhancement, their position was varied relative to several artificial promoter-gene combinations. It was found that SARs enhance general promoter functions in an orientation- and partially distance-independent manner; their effect is restricted to the integrated state of transfected templates. During the phase of transient expression, SAR elements were generally found to have an antagonizing effect.