Analysis of the chromatin domain organisation around the plastocyanin gene reveals an MAR-specific sequence element in Arabidopsis thaliana.

The Arabidopsis thaliana genome is currently being sequenced, eventually leading towards the unravelling of all potential genes. We wanted to gain more insight into the way this genome might be organized at the ultrastructural level. To this extent we identified matrix attachment regions demarking potential chromatin domains, in a 16 kb region around the plastocyanin gene. The region was cloned and sequenced revealing six genes in addition to the plastocyanin gene. Using an heterologous in vitro nuclear matrix binding assay, to search for evolutionary conserved matrix attachment regions (MARs), we identified three such MARs. These three MARs divide the region into two small chromatin domains of 5 kb, each containing two genes. Comparison of the sequence of the three MARs revealed a degenerated 21 bp sequence that is shared between these MARs and that is not found elsewhere in the region. A similar sequence element is also present in four other MARs of Arabidopsis.Therefore, this sequence may constitute a landmark for the position of MARs in the genome of this plant. In a genomic sequence database of Arabidopsis the 21 bp element is found approximately once every 10 kb. The compactness of the Arabidopsis genome could account for the high incidence of MARs and MRSs we observed.     

Structural domains and matrix attachment regions along colinear chromosomal segments of maize and sorghum

Although a gene's location can greatly influence its expression, genome sequencing has shown that orthologous genes may exist in very different environments in the genomes of closely related species. Four genes in the maize alcohol dehydrogenase (adh1) region represent solitary genes dispersed among large repetitive blocks, whereas the orthologous genes in sorghum are located in a different setting surrounded by low-copy-number DNAs. A specific class of DNA sequences, matrix attachment regions (MARs), was found to be in comparable positions in the two species, often flanking individual genes. If these MARs define structural domains, then the orthologous genes in maize and sorghum should experience similar chromatin environments. In addition, MARs were divided into two groups, based on the competitive affinity of their association with the matrix. The "durable" MARs retained matrix associations at the highest concentrations of competitor DNA. Most of the durable MARs mapped outside genes, defining the borders of putative chromatin loops. The "unstable" MARs lost their association with the matrix under similar competitor conditions and mapped mainly within introns. These results suggest that MARs possess both domain-defining and regulatory roles. Miniature inverted repeat transposable elements (MITEs) often were found on the same fragments as the MARs. Our studies showed that many MITEs can bind to isolated nuclear matrices, suggesting that MITEs may function as MARs in vivo.     

Matrix attachment regions and structural colinearity in the genomes of two grass species.

In order to gain insights into the relationship between spatial organization of the genome and genome function we have initiated studies of the co-linear Sh2/A1- homologous regions of rice (30 kb) and sorghum (50 kb). We have identified the locations of matrix attachment regions (MARs) in these homologous chromosome segments, which could serve as anchors for individual structural units or loops. Despite the fact that the nucleotide sequences serving as MARs were not detectably conserved, the general organizational patterns of MARs relative to the neighboring genes were preserved. All identified genes were placed in individual loops that were of comparable size for homologous genes. Hence, gene composition, gene orientation, gene order and the placement of genes into structural units has been evolutionarily conserved in this region. Our analysis demonstrated that the occurrence of various 'MAR motifs' is not indicative of MAR location. However, most of the MARs discovered in the two genomic regions were found to co-localize with miniature inverted repeat transposable elements (MITEs), suggesting that MITEs preferentially insert near MARs and/or that they can serve as MARs.     

A global but stable change in HeLa cell morphology induces reorganization of DNA structural loop domains within the cell nucleus

DNA of higher eukaryotes is organized in supercoiled loops anchored to a nuclear matrix (NM). The DNA loops are attached to the NM by means of non-coding sequences known as matrix attachment regions (MARs). Attachments to the NM can be subdivided in transient and permanent, the second type is considered to represent the attachments that subdivide the genome into structural domains. As yet very little is known about the factors involved in modulating the MAR-NM interactions. It has been suggested that the cell is a vector field in which the linked cytoskeleton-nucleoskeleton may act as transducers of mechanical information. We have induced a stable change in the typical morphology of cultured HeLa cells, by chronic exposure of the cells to the polar compound dimethylsulfoxide (DMSO). Using a PCR-based method for mapping the position of any DNA sequence relative to the NM, we have monitored the position relative to the NM of sequences corresponding to four independent genetic loci located in separate chromosomes representing different territories within the cell nucleus. Here, we show that stable modification of the NM morphology correlates with the redefinition of DNA loop structural domains as evidenced by the shift of position relative to the NM of the c-myc locus and the multigene locus PRM1 --> PRM2 --> TNP2, suggesting that both cell and nuclear shape may act as cues in the choice of the potential MARs that should be attached to the NM.     

Why repetitive DNA is essential to genome function

There are clear theoretical reasons and many well-documented examples which show that repetitive, DNA is essential for genome function. Generic repeated signals in the DNA are necessary to format expression of unique coding sequence files and to organise additional functions essential for genome replication and accurate transmission to progeny cells. Repetitive DNA sequence elements are also fundamental to the cooperative molecular interactions forming nucleoprotein complexes. Here, we review the surprising abundance of repetitive DNA in many genomes, describe its structural diversity, and discuss dozens of cases where the functional importance of repetitive elements has been studied in molecular detail. In particular, the fact that repeat elements serve either as initiators or boundaries for heterochromatin domains and provide a significant fraction of scaffolding/matrix attachment regions (S/MARs) suggests that the repetitive component of the genome plays a major architectonic role in higher order physical structuring. Employing an information science model, the 'functionalist' perspective on repetitive DNA leads to new ways of thinking about the systemic organisation of cellular genomes and provides several novel possibilities involving repeat elements in evolutionarily significant genome reorganisation. These ideas may facilitate the interpretation of comparisons between sequenced genomes, where the repetitive DNA component is often greater than the coding sequence component.     

Possible role of H1 histone in replication timing

AT‐rich repetitive DNA sequences become late replicating during cell differentiation. Replication timing is not correlated with LINE density in human cells (Ryba et al. 2010). However, short and properly spaced runs of oligo dA or dT present in nuclear matrix attachment regions (MARs) of the genome are good candidates for elements of AT‐rich repetitive late replicating DNA. MAR attachment to the nuclear matrix is negatively regulated by chromatin binding of H1 histone, but this is counteracted by H1 phosphorylation, high mobility group proteins or, indirectly, core histone acetylation. Fewer MAR attachments correlates positively with longer average DNA loop size, longer replicons and an increase of late replicating DNA.     

A map of nuclear matrix attachment regions within the breast cancer loss-of-heterozygosity region on human chromosome 16q22.1

There is abundant evidence that the DNA in eukaryotic cells is organized into loop domains that represent basic structural and functional units of chromatin packaging. To explore the DNA domain organization of the breast cancer loss-of-heterozygosity region on human chromosome 16q22.1, we have identified a significant portion of the scaffold/matrix attachment regions (S/MARs) within this region. Forty independent putative S/MAR elements were assigned within the 16q22.1 locus. More than 90% of these S/MARs are AT rich, with GC contents as low as 27% in 2 cases. Thirty-nine (98%) of the S/MARs are located within genes and 36 (90%) in gene introns, of which 15 are in first introns of different genes. The clear tendency of S/MARs from this region to be located within the introns suggests their regulatory role. The S/MAR resource constructed may contribute to an understanding of how the genes in the region are regulated and of how the structural architecture and functional organization of the DNA are related.     

A matrix/scaffold attachment region binding protein: identification, purification, and mode of binding.

Matrix/scaffold attachment regions (MARs/SARs) partition chromatin into functional loop domains. Here we have identified a chicken protein that selectively binds to MARs from the chicken lysozyme locus and to MARs from Drosophila, mouse, and human genes. This protein, named ARBP (for attachment region binding protein), was purified to homogeneity and shown to bind to MARs in a cooperative fashion. ARBP is an abundant nuclear protein and a component of the internal nuclear network. Deletion mutants indicate that multiple AT-rich sequences, if contained in a minimal approximately 350 bp MAR fragment, can lead to efficient binding of ARBP. Furthermore, dimerization mutants show that, to bind ARBP efficiently, MAR sequences can act synergistically over large distances, apparently with the intervening DNA looping out. The binding characteristics of ARBP to MARs reproduce those of unfractionated matrix preparations, suggesting that ARBP is an important nuclear element for the generation of functional chromatin loops.     

Unichrom, a novel nuclear matrix protein, binds to the Ars insulator and canonical MARs

Eukaryotic genomic DNA is organized into loop structures by attachments to the nuclear matrix. These attachments to the nuclear matrix have been supposed to form the boundaries of chromosomal DNA. Insulators or boundary elements are defined by two characteristics: they interrupt promoter-enhancer communications when inserted between them, and they suppress the silencing of transgenes stably integrated into inactive chromosomal domains. We recently identified an insulator element in the upstream region of the sea urchin arylsulfatase (HpArs) gene that shows both enhancer blocking and suppression of position effects. Here, we report that Unichrom, originally identified by its G-stretch DNA binding capability, is a nuclear matrix protein that binds to the Ars insulator and canonical nuclear matrix attachment regions (MARs). We also show that Unichrom recognizes the minor groove of the AT-rich region within the Ars insulator, which may have a base-unpairing property, as well as the G-stretch DNA. Furthermore, Unichrom selectively interacts with poly(dG).poly(dC), poly(dA).poly(dT) and poly(dAT).poly(dAT), but not with poly(dGC).poly(dGC). Unichrom also shows high affinity for single-stranded G- and C-stretches. We discuss the DNA binding motif of Unichrom and the function of Unichrom in the nuclear matrix.     

Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites

Introduction of torsional stress into active chromatin domains requires that linear DNA molecules be anchored in vivo to impede free rotation. While searching for these anchorage elements, we have localized a nuclear matrix association region (MAR) within the mouse immunoglobulin kappa gene that contains two topoisomerase II sites and is adjacent to the tissue-specific enhancer. The same matrix contact occurs when the kappa locus is in germ-line (inactive) or rear-ranged (transcribed) configurations. This constitutive anchorage site partitions the gene into V-J and C region chromatin domains. We demonstrate that at least 10,000 similar and evolutionarily conserved MAR binding sites exist in the nucleus. We propose that these sites, in association with topoisomerase II and possibly in conjunction with enhancers, play fundamental roles in the functional organization of chromatin loop domains.     

Computational and in vitro analysis of destabilized DNA regions in the interferon gene cluster: potential of predicting functional gene domains

Recent evidence adds support to a traditional concept according to which the eukaryotic nucleus is organized into functional domains by scaffold or matrix attachment regions (S/MARs). These regions have previously been predicted to have a high potential for stress-induced duplex destabilization (SIDD). Here we report the parallel results of binding (reassociation) and computational SIDD analyses for regions within the human interferon gene cluster on the short arm of chromosome 9 (9p22). To verify and further refine the biomathematical methods, we focus on a 10 kb region in the cluster with the pseudogene IFNWP18 and the interferon alpha genes IFNA10 and IFNA7. In a series of S/MAR binding assays, we investigate the promoter and termination regions and additional attachment sequences that were detected in the SIDD profile. The promoters of the IFNA10 and the IFNA7 genes have a moderate approximately 20% binding affinity to the nuclear matrix; the termination sequences show stronger association (70-80%) under our standardized conditions. No comparable destabilized elements were detected flanking the IFNWP18 pseudogene, suggesting that selective pressure acts on the physicochemical properties detected here. In extended, noncoding regions a striking periodicity is found of rather restricted SIDD minima with scaffold binding potential. By various criteria, the underlying sequences represent a new class of S/MARs, thought to be involved in a higher level organization of the genome. Together, these data emphasize the relevance of SIDD calculations as a valid approach for the localization of structural, regulatory, and coding regions in the eukaryotic genome.     

Novel functional MAR elements of double minute chromosomes in human ovarian cells capable of enhancing gene expression

Double minute chromosomes or double minutes (DMs) are cytogenetic hallmarks of extrachromosomal genomic amplification and play a critical role in tumorigenesis. Amplified copies of oncogenes in DMs have been associated with increased growth and survival of cancer cells but DNA sequences in DMs which are mostly non-coding remain to be characterized. Following sequencing and bioinformatics analyses, we have found 5 novel matrix attachment regions (MARs) in a 682 kb DM in the human ovarian cancer cell line, UACC-1598. By electrophoretic mobility shift assay (EMSA), we determined that all 5 MARs interact with the nuclear matrix in vitro. Furthermore, qPCR analysis revealed that these MARs associate with the nuclear matrix in vivo, indicating that they are functional. Transfection of MARs constructs into human embryonic kidney 293T cells showed significant enhancement of gene expression as measured by luciferase assay, suggesting that the identified MARS, particularly MARs 1 to 4, regulate their target genes in vivo and are potentially involved in DM-mediated oncogene activation.