Episomal minicircles persist in periods of transcriptional inactivity and can be transmitted through somatic cell nuclear transfer into bovine embryos.

Episomal plasmids based on a scaffold/matrix attachment region (S/MAR) are extrachromosomal DNA entities that replicate once per cell cycle and are stably maintained in cells or tissue. We generated minicircles, episomal plasmids devoid of bacterial sequences, and show that they are stably transmitted in clonal primary bovine fibroblasts without selection pressure over more than two months. Total DNA, plasmid extraction and fluorescence in situ hybridization (FISH) analyses suggest that the minicircles remained episomal and were not integrated into the genome. Minicircles survived extended periods in serum-starved cells, which indicates that ongoing transcription in non-proliferating cells is not necessary for the maintenance of S/MAR-episomes. To test whether minicircles endure the process of somatic cell nuclear transfer (SCNT), we used cell-cycle synchronized, serum-starved, minicircle-containing cells. Analysis of cells outgrown from SCNT-derived blastocysts shows that the minicircles are maintained through SCNT and early embryonic development, which raises the prospect of using cell lines with episomal minicircles for the generation of transgenic animals.

Cytoplasmic injection of circular plasmids allows targeted expression in mammalian embryos.

Injection of linearized DNA constructs into the pronuclei of fertilized mammalian eggs is a standard method for producing transgenic embryos and animals. Here, we show that injection of covalently closed circular (ccc) plasmids into the cytoplasm of fertilized bovine and murine eggs is a highly efficient and simple alternative for ectopic expression of foreign DNA in embryos. A broad range of plasmids could be successfully expressed in preimplantation stages, including plasmids and minicircles with a scaffold/matrix attachment region (S/MAR), conventional plasmids, and bacterial artificial chromosomes (BACs). Although the foreign DNA plasmids are mainly maintained as episomal entities during preimplantation development, they accurately behave like nuclear DNA. Onset of transcription of an Oct4 promoter-controlled marker gene coincided with the species-specific time points of major embryonic genome activation, and could be modulated by in vitro DNA-methylation. This approach allows an experimental access to reprogramming events in early mammalian embryos.

Identification, genome mapping, and CTCF binding of potential insulators within the FXYD5-COX7A1 locus of human chromosome 19q13.12.

Identification of insulators is one of the most difficult problems in functional mapping of genomes. For this reason, up to now only a few insulators have been described. In this article we suggest an approach that allows direct isolation of insulators by a simple positive-negative selection based on blocking enhancer effects by insulators. The approach allows selection of fragments capable of blocking enhancers from mixtures of genomic fragments prepared from up to 1-Mb genomic regions. Using this approach, a 1-Mb human genome locus was analyzed and eight potential insulators were selected. Five of the eight sequences were positioned in intergenic regions and two were within introns. The genes of the alpha-polypeptide H+/K+ exchanging ATPase (ATP4A) and amyloid beta (A4) precursor-like protein 1 (APLP1) within the locus studied were found to be flanked by insulators on both sides. Both genes are characterized by distinct tissue-specific expression that differs from the tissue specificity of the surrounding genes. The data obtained are consistent with the conception that insulators subdivide genomic DNA into loop domains that comprise genes characterized by similar expression profiles. Using chromatin immunoprecipitation assay, we demonstrated also that at least six of the putative insulators revealed in this work could bind the CTCF transcription factor in vivo. We believe that the proposed approach could be a useful instrument for functional analysis of genomes.

Correlations between scaffold/matrix attachment region (S/MAR) binding activity and DNA duplex destabilization energy.

Scaffold or matrix-attachment regions (S/MARs) are thought to be involved in the organization of eukaryotic chromosomes and in the regulation of several DNA functions. Their characteristics are conserved between plants and humans, and a variety of biological activities have been associated with them. The identification of S/MARs within genomic sequences has proved to be unexpectedly difficult, as they do not appear to have consensus sequences or sequence motifs associated with them. We have shown that S/MARs do share a characteristic structural property, they have a markedly high predicted propensity to undergo strand separation when placed under negative superhelical tension. This result agrees with experimental observations, that S/MARs contain base-unpairing regions (BURs). Here, we perform a quantitative evaluation of the association between the ease of stress-induced DNA duplex destabilization (SIDD) and S/MAR binding activity. We first use synthetic oligomers to investigate how the arrangement of localized unpairing elements within a base-unpairing region affects S/MAR binding. The organizational properties found in this way are applied to the investigation of correlations between specific measures of stress-induced duplex destabilization and the binding properties of naturally occurring S/MARs. For this purpose, we analyze S/MAR and non-S/MAR elements that have been derived from the human genome or from the tobacco genome. We find that S/MARs exhibit long regions of extensive destabilization. Moreover, quantitative measures of the SIDD attributes of these fragments calculated under uniform conditions are found to correlate very highly (r2>0.8) with their experimentally measured S/MAR-binding strengths. These results suggest that duplex destabilization may be involved in the mechanisms by which S/MARs function. They suggest also that SIDD properties may be incorporated into an improved computational strategy to search genomic DNA sequences for sites having the necessary attributes to function as S/MARs, and even to estimate their relative binding strengths.

Performance of genomic bordering elements at predefined genomic loci.

Eukaryotic DNA is organized into chromatin domains that regulate gene expression and chromosome behavior. Insulators and/or scaffold-matrix attachment regions (S/MARs) mark the boundaries of these chromatin domains where they delimit enhancing and silencing effects from the outside. By recombinase-mediated cassette exchange (RMCE), we were able to compare these two types of bordering elements at a number of predefined genomic loci. Flanking an expression vector with either S/MARs or two copies of the non-S/MAR chicken hypersensitive site 4 insulator demonstrates that while these borders confer related expression characteristics at most loci, their effect on chromatin organization is clearly distinct. Our results suggest that the activity of bordering elements is most pronounced for the abundant class of loci with a low but negligible expression potential in the case of highly expressed sites. By the RMCE procedure, we demonstrate that expression parameters are not due to a potential targeting action of bordering elements, in the sense that a linked transgene is directed into a special class of loci. Instead, we can relate the observed transcriptional augmentation phenomena to their function as genomic insulators.

Poly(ADP-ribose) polymerase-1: association with nuclear lamins in rodent liver cells.

The distribution of poly(ADP-ribose) polymerase-1 (PARP-1) over different nuclear compartments was studied by nuclear fractionation procedures and Western analysis revealing a prominent role of the nuclear matrix. This structure is operationally defined by the solubility properties of the A- and B-type lamins under defined experimental conditions. We consistently observed that most of the nuclear matrix-associated PARP-1 partitioned, in an active form, with the insoluble, lamin-enriched protein fractions that were prepared by a variety of established biochemical procedures. These PARP-1-protein interactions resisted salt extraction, disulfide reduction, RNase and DNase digestion. An inherent ability of PARP-1 to reassemble with the lamins became evident after a cycle of solubilization/dialysis using either urea or Triton X-100 and disulfide reduction, indicating that these interactions were dominated by hydrophobic forces. Together with in vivo crosslinking and co-immunoprecipitation experiments our results show that the lamins are prominent PARP-1-binding partners which could contribute to the functional sequestration of the enzyme on the nuclear matrix.

Nuclear scaffold/matrix attached region modules linked to a transcription unit are sufficient for replication and maintenance of a mammalian episome.

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 scaffold/matrix 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 scaffold/matrix 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.

Elk-1 knock-out mice engineered by Flp recombinase-mediated cassette exchange.

Elk-1 is a member of the TCF subfamily of Ets proteins. TCFs interact with SRF at serum response elements (SREs) of immediate early genes (IEGs), such as c-fos and Egr-1, thereby mediating IEG induction upon extracellular stimulation. We previously generated an Elk-1 null allele (Elk1-137) in murine embryonic stem (ES) cells by homologous recombination. In Elk1-137, the Elk-1 gene was replaced by a Hygromycin B phosphotransferase - Thymidine Kinase (HygTk) fusion gene, flanked by two nonidentical Flp recombinase recognition (FRT) sites (Cesari et al., [2004] Mol Cell Biol, in press) to allow for the subsequent generation of alternative alleles of interest by recombinase-mediated cassette exchange (RMCE). Elk1-deficient mice derived from Elk-1((137/0)) ES cells are viable and do not reveal strong phenotypical abnormalities, apart from male sterility. However, the Elk-1 locus contains the Tk cassette, which has previously been related to this defect. Therefore, in our first experiment involving the technique of Flp RMCE we chose to remove the HygTk cassette in Elk-1((137/0)) ES cells and to generate Elk-1((RMCE16/0)) and Elk-1((RMCE16/RMCE16)) mice. In so doing, we provide evidence that the sterility of Elk1((137/0)) mice was not due to the absence of Elk-1 but rather the presence of HygTk. This is the first report of mice derived from ES cells which were subjected to Flp RMCE and thus proves that RMCE is a powerful tool for the genetic engineering of previously tagged loci in the mouse genome.

Molecular structure and regulatory potential of a T-DNA integration site in petunia.

The genomic structure surrounding a T-DNA integration site in a transgenic petunia plant, which shows deregulation of a root-specific promoter, was investigated. We have already demonstrated that T-DNA integration in this transformant (P13) had occurred close to a scaffold/matrix attachment region (S/MAR). A major question regarding the observed promoter leakiness was whether the T-DNA had integrated into the centre or at the border of the Petun-SAR and whether other regulatory elements are located within this genomic region. While small rearrangements were shown to occur during T-DNA integration in agreement with other reports, we find indications of the presence of a SINE retroposon--an apparent landmark for recombinogenic targets--at the integration site. Binding assays to both plant and animal nuclear scaffolds, supported by biomathematical analyses, reveal that the T-DNA is definitely located at the border of a strong S/MAR, which is in agreement with current models on the structure of integration sites. These results, together with a developmentally regulated leaf-specific enhancer effect of the Petun-SAR on gene expression in transgenic tobacco plants, indicate that the Petun-SAR demarcates the right border of a chromatin domain with genes predominantly active in leaves.

An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo.

pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA-protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.

Multiple collagen I gene regulatory elements have sites of stress-induced DNA duplex destabilization and nuclear scaffold/matrix association potential.

The availability of the complete nucleotide sequences of numerous prokaryotic and eukaryotic organisms should stimulate the development and application of computer-based approaches for studying genome organization and function. Earlier work has shown that distinct regulatory DNA elements can be identified by computational analysis as sites of stress-induced DNA duplex destabilization (SIDD). Here we report the results of computational and experimental analyses of previously identified regulatory elements in the murine alpha1(I) collagen (Col1a1) gene domain. We found that several distal 5' DNase I-hypersensitive sites (HSs) which function in the chromatin loop organization of the Col1a1 gene are characterized by strongly destabilized SIDD profiles. Elements in the proximal 5' promoter and first intron which differentially regulate Col1a1 promoter activity in different collagen-producing cell types also contain SIDD sites. All 5' elements associated with destabilized sites are shown to have nuclear matrix binding activity in an in vitro binding assay. Other putative regulatory elements in the transcribed and 3'-flanking regions of the Col1a1 gene, including both of its polyadenylation sites, are also associated with SIDD peaks. The human COL1A1 gene has periodic SIDD peaks within the transcribed region, suggesting that abundantly expressed genes may require SIDDs acting as topological sinks during transcription. The 5' ends of the murine Col1a1 and the homologous human gene revealed similar SIDD profiles, but limited DNA sequence similarity, indicating that some DNA functions are evolutionarily conserved by preserving higher order DNA structural properties rather than nucleotide sequence. Our results show that destabilized SIDD profiles are a common feature of eukaryotic regulatory DNA elements with such diverse functions as chromatin organization, cell-specific transcriptional enhancement, and initiation and termination of transcription. They demonstrate the usefulness of computational analyses that predict SIDD properties in reliably identifying DNA elements involved in the structural organization of the eukaryotic genome and the regulation of its expression.

S/MARt DB: a database on scaffold/matrix attached regions.

S/MARt DB, the S/MAR transaction database, is a relational database covering scaffold/matrix attached regions (S/MARs) and nuclear matrix proteins that are involved in the chromosomal attachment to the nuclear scaffold. The data are mainly extracted from original publications, but a World Wide Web interface for direct submissions is also available. S/MARt DB is closely linked to the TRANSFAC database on transcription factors and their binding sites. It is freely accessible through the World Wide Web (http://transfac.gbf.de/SMARtDB/) for non-profit research.