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Results for “Matrix Attachment Regions”

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PubMed2002

The function of the nuclear matrix attachment region of silkworm rDNA as an autonomously replicating sequence in plasmid and chromosomal replication origin in yeast.

Nuclear matrix attachment regions (MARs) play a crucial role in chromatin architecture, gene expression, and DNA replication. Although it is well known that yeast autonomously replicating sequences (ARSs) bind nuclear matrix and MARs also function as ARS elements in yeast, whether a heterologous MAR or ARS element acts as a replication origin in the chromosome has not been elucidated. We previously identified a MAR (rMAR) located in the nontranscribed spacer (NTS) of silkworm Attacus ricini rDNA. We report here that this rMAR contains 10 copies of ARS consensus sequence (ACS) and several DNA unwinding regions. The rMAR employs ARS activity in yeast and a rARS element locates in the 3(') region of the rMAR. Furthermore, we have also revealed that either the rMAR or the rARS element functions as a replication origin in the chromosome. Our results provide the first direct evidence to demonstrate that heterologous rMAR and rARS display chromosomal origin activity, suggesting that the chromosome structure and replication origin of rDNA reserve some common features during evolution.

Animals↗

PubMed2005

The rb7 matrix attachment region increases the likelihood and magnitude of transgene expression in tobacco cells: a flow cytometric study.

Many studies in both plant and animal systems have shown that matrix attachment regions (MARs) can increase expression of transgenes in whole organisms or cells in culture. Because histochemical assays often indicate variegated transgene expression, a question arises: Do MARs increase transgene expression by increasing the percentage of cells expressing the transgene (likelihood), by increasing the level of expression in expressing cells (magnitude), or both? To address this question, we used flow cytometry to measure green fluorescent protein (GFP) expression in individual tobacco (Nicotiana tabacum) cells from lines transformed by Agrobacterium tumefaciens. We conclude that MAR-mediated overall increases in transgene expression involve both likelihood and magnitude. On average, cell lines transformed with the Rb7 MAR-containing vector expressed GFP at levels 2.0- to 3.7-fold higher than controls. MAR lines had fewer nonexpressing cells than control lines (10% versus 45%), and the magnitude of GFP expression in expressing cells was greater in MAR lines by 1.9- to 2.9-fold. We also show that flow cytometry measurements on cells from isogenic lines are consistent with those from populations of independently transformed cell lines. By obviating the need to establish isogenic lines, this use of flow cytometry could greatly simplify the evaluation of MARs or other sequence elements that affect transgene expression.

Agrobacterium tumefaciens↗

PubMed2000

Modulated binding of SATB1, a matrix attachment region protein, to the AT-rich sequence flanking the major breakpoint region of BCL2.

The t(14,18) chromosomal translocation that occurs in human follicular lymphoma constitutively activates the BCL2 gene and disrupts control of apoptosis. Interestingly, 70% of the t(14,18) translocations are confined to three 15-bp clusters positioned within a 150-bp region (major breakpoint region or [MBR]) in the untranslated portion of terminal exon 3. We analyzed DNA-protein interactions in the MBR, as these may play some role in targeting the translocation to this region. An 87-bp segment (87MBR) immediately 3' to breakpoint cluster 3 was essential for DNA-protein interaction monitored with mobility shift assays. We further delineated a core binding region within 87MBR: a 33-bp, very AT-rich sequence highly conserved between the human and mouse BCL2 gene (37MBR). We have purified and identified one of the core factors as the matrix attachment region (MAR) binding protein, SATB1, which is known to bind to AT-rich sequences with a high propensity to unwind. Additional factors in nuclear extracts, which we have not yet characterized further, increased SATB1 affinity for the 37MBR target four- to fivefold. Specific binding activity within 37MBR displayed cell cycle regulation in Jurkat T cells, while levels of SATB1 remained constant throughout the cell cycle. Finally, we demonstrated in vivo binding of SATB1 to the MBR, strongly suggesting the BCL2 major breakpoint region is a MAR. We discuss the potential consequences of our observations for both MBR fragility and regulatory function.

Amino Acid Sequence↗

PubMed2005

Nuclear matrices and matrix attachment regions from Green alga: Dunaliella salina.

Nuclear DNA of eukaryotic organism attaches to the proteinaceous nuclear matrices via specific matrix attachment regions (MARs). In order to investigate the interactions between chromosomal DNA and nuclear matrices,we isolated the MARs from unicellular alga Dunaliella salina. As the first step,a random MAR library was set up and then the binding affinity of the selected clones to nuclear matrices was tested in this study. Three DNA fragments were found to bind specifically to the nuclear matrices in vitro,of which two were strong binders and all contained known consensus motifs and a hairpin loop structure of MAR.

Base Sequence↗

PubMed1999

Matrix attachment region binding protein MFP1 is localized in discrete domains at the nuclear envelope.

Recently, it has been suggested that nuclear processes, such as replication, transcription, and splicing, are spatially organized and associated with a nuclear framework called the nuclear matrix, a structure of unknown molecular composition. It has been shown that chromatin is attached to the nuclear matrix via specific DNA fragments called matrix attachment regions (MARs). We have begun to dissect the plant nuclear matrix by isolating a DNA binding protein with specific affinity for MARs. Here, it is shown that MAR binding filament-like protein 1 (MFP1) is associated with specklelike structures at the nuclear periphery that are part of isolated nuclei and the nuclear matrix. A predicted N-terminal transmembrane domain is necessary for the specific targeting of MFP1 to the speckles, indicating an association with the nuclear envelope-endoplasmic reticulum continuum. In addition, it is shown that a marker protein for plant microtubule organizing centers, which has been shown to be localized on the outside of the plant nuclear envelope, is also part of the nuclear matrix. These findings indicate a close and previously undescribed connection in plants between the nuclear envelope and the internal nuclear matrix, and they suggest a function for MFP1 in attaching chromatin to specific sites at the nuclear periphery.

Arabidopsis Proteins↗

PubMed1996

Dissection of the ability of the chicken lysozyme gene 5' matrix attachment region to stimulate transgene expression and to dampen position effects.

The chicken lysozyme gene domain is flanked by nuclear matrix attachment regions (MARs) on each side. We have previously shown that bilaterally flanking 5'MARs in stably transfected artificial genetic units enhance expression of a reporter transgene and dampen position effects of the chromatin structure at the site of integration. The 5' MAR was now dissected into smaller fragments that were monitored for effects on transgene expression in mouse 3T3 cells by a similar assay. Fragments, which contain 1.32 and 1.45 kb and represent the upstream and the downstream half, respectively, of the 5' MAR, retained the ability to stimulate transgene expression as well as the ability to reduce the variation in the level of expression. However, a 452 bp subfragment (H1-HaeII), which still exhibits specific binding to nuclear matrices and contains two high-affinity binding sites for the abundant nuclear matrix protein ARBP, lost both of those abilities. A dimerized 177 bp sequence from fragment H1-HaeII, which also binds selectively to nuclear matrices and includes a duplicated ARBP binding site, was also unable to stimulate reporter gene expression. Furthermore, a 0.65 kb subfragment containing an intrinsically bent sequence did not affect an elevated reporter gene expression and its dampening. Our results show that the ability of MAR fragments to bind to nuclear matrices is not sufficient to enhance and insulate transgene expression in stably transfected cells.

3T3 Cells↗

PubMed1996

Contrasting effects of the SATB1 core nuclear matrix attachment region and flanking sequences of the keratin 18 gene in transgenic mice.

The 2.3 kb and 3.5 kb of DNA that flank the human keratin 18 (K18) gene and synthetic nuclear matrix attachment regions (MAR) composed of the binding sites for the SATB1 nuclear protein were fused to a reporter gene that utilizes the mouse metallothionein promoter and the human growth hormone gene (MThGH). Transgenic mice were generated from both constructions and the control MThGH gene to test K18 and SATB1 MAR sequences for the ability to insulate the reporter gene from integration site-specific position effects. The MThGH control gene was variably expressed in brain, heart, intestine, kidney, liver, and testes, confirming previous studies. In contrast, the MThGH gene insulated by the K18 flanking sequences was expressed in the same tissues of four independent transgenic animals at levels correlated with the copy number except for intestine. The average level of expression on a per gene basis of the K18 insulated gene was from 9- to 49-fold higher than the control. The MThGH gene linked to the SATB1 MAR sequences was completely repressed in the brains and kidneys of all six transgenic mice. However, expression was nearly as efficient in testes as the K18-insulated gene. Both the SATB1 MAR and the K18 flanking sequences confer position-independent transcriptional status on the reporter gene in some or many tissues. However, the effects are stimulatory for the K18 elements and generally suppressive for the SATB1 MAR elements.

Animals↗

PubMed2006

Selective matrix attachment regions in T helper cell subsets support loop conformation in the Ifng gene.

Cytokine genes undergo progressive changes in chromatin organization when naïve CD4+ T helper (Th) cells differentiate into committed Th1 and Th2 lineages. Here, we analyzed nuclear matrix attachment regions (MARs) in the Ifng gene by DNA array technique in unactivated and activated CD4+ Th cells. This approach was combined with analysis of spatial organization of the Ifng gene by chromosome conformation capture approach to assess the relationship between the gene conformation and matrix attachment organization in functionally different cell subsets. We report that the Ifng gene in unactivated cells displays a linear conformation, but in T-cell receptor-activated cells, it adopts a loop conformation. The selective MARs support the spatial gene organization and characteristically define the Ifng gene in functionally different cell subsets. The pattern of interaction of the Ifng gene with the nuclear matrix dynamically changes in a lineage-specific manner in parallel with the changes in Ifng gene conformation. The data suggest that such structural dynamics provide the means for transcriptional regulation of the Ifng gene in the course of activation and differentiation of CD4+Th cells.

Animals↗

PubMed1992

Partial characterization of nuclear matrix attachment regions from human fibroblast DNA using Alu-polymerase chain reaction.

The proteinaceous nuclear matrix of mammalian cell nuclei has been suggested to be involved in the regulation of chromatin structure, DNA replication, and gene expression. Interaction between cellular DNA and the nuclear matrix is mediated by putative DNA binding sequences, matrix attachment regions (MARs), which may become altered during early events in cellular transformation. Among the cellular changes occurring during the development of neoplasia, all of which may potentially involve the nuclear matrix, are alterations in nuclear structure, loss of control of DNA replication, and significant modifications of cellular gene expression. Therefore, a better understanding of the interaction between DNA and the nuclear matrix is needed. Isolated matrix associated DNA from pulse labeled SV40 transformed human fibroblasts was shown to be enriched in newly replicated DNA, confirming the association of DNA replication with the nuclear matrix as observed by others. Subgenomic fractions of matrix associated DNA enriched in putative MARs sites were prepared from quiescent and logarithmically growing normal human fibroblasts and SV40 transformed human fibroblasts. These fractions of DNA were analyzed by Alu-polymerase chain reaction and agarose gel electrophoresis, revealing complex and unique patterns of DNA products for each cell type investigated. A number of prominent DNA fragments with similar molecular size were found to be present in the amplified DNA products of each DNA source, suggesting that these DNA fragments may represent common DNA sequences which contain MARs sites or which are associated with MARs sites. The application of Alu-polymerase chain reaction to the molecular analysis of nuclear matrix associated DNA may facilitate the isolation and characterization of potentially new human MARs sequences.

DNA↗

PubMed2004

Large-scale identification and mapping of nuclear matrix-attachment regions in the distal imprinted domain of mouse chromosome 7.

Mammalian imprinted genes, which are expressed from only one of the parental alleles, have a tendency to form clusters and are regulated by long-range mechanisms. Nuclear matrix-attachment regions (MARs), the anchor points of loop domains, are involved in coordination of gene expression and could play a role in regulation of imprinted domains. We have identified and mapped a total of 52 MARs in a 1-Mb imprinted domain on mouse distal chromosome 7 using our cosmid contigs and an in vitro MAR assay. We find two MAR clusters (comprising 20 and 19 MARs), one of which is mapped in the Th-Ins2 intergenic region, coincident with the boundary between the two imprinted subdomains. However, the imprinted/non-imprinted boundaries are not associated with a MAR. Based on the sequence information, we find that many of the MARs are rich in long interspersed nuclear elements. In addition, comparisons of the results obtained with several MAR-prediction software programs reveal good performance of ChrClass in terms of both sensitivity and specificity. This study presents the first large-scale mapping of MARs in an imprinted domain and provides a platform for understanding the roles of MARs in imprinting.

Animals↗

PubMed1999

Flanking nuclear matrix attachment regions synergize with the T cell receptor delta enhancer to promote V(D)J recombination.

Previous studies have identified nuclear matrix attachment regions (MARs) that are closely associated with transcriptional enhancers in the IgH, Igkappa, and T cell receptor (TCR) beta loci, but have yielded conflicting information regarding their functional significance. In this report, a combination of in vitro and in situ mapping approaches was used to localize three MARs associated with the human TCR delta gene. Two of these are located within the Jdelta3-Cdelta intron, flanking the core TCR delta enhancer (Edelta) both 5' and 3' in a fashion reminiscent of the Ig heavy chain intronic enhancer-associated MARs. The third is located about 20 kb upstream, tightly linked to Ddelta1 and Ddelta2. We have previously used a transgenic minilocus V(D)J recombination reporter to establish that Edelta functions as a developmental regulator of V(D)J recombination, and that it does so by modulating substrate accessibility to the V(D)J recombinase. We show here that the Edelta-associated MARs function synergistically with the core Edelta to promote V(D)J recombination in this system, as they are required for enhancer-dependent transgene rearrangement in single-copy transgene integrants.

Animals↗

PubMed1990

The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes.

Matrix attachment regions (MARs) are DNA elements that dissect the genome into topologically separated domains by binding to a chromosomal skeleton. This study explored the putative influence of the MAR located 5' of the chicken lysozyme gene on expression of heterologous genes in heterologous cell systems. Expression of a construct with the chloramphenicol acetyltransferase (CAT) indicator gene controlled by the herpes simplex virus thymidine kinase promoter (TC) and a construct in which the same transcriptional unit is flanked by chicken lysozyme 5' MARs (MTCM) was assayed after stable transfection into rat fibroblasts. Median CAT activity per copy number in MTCM transfectants was elevated approximately 10-fold relative to that in TC transfectants. Total variation in normalized CAT activity decreased from more than 100-fold among TC transfectants to nearly 6-fold among MTCM transfectants. The steady-state level of transcripts and the relative rate of transcription were increased in MTCM transfectants, as shown by S1 nuclease and run-on transcription assays, respectively. The chicken lysozyme 5' MAR thus can confer elevated, less position-dependent expression on a heterologous promoter in cells of a different species by increasing the density of transcribing RNA polymerase molecules. MAR-mediated transcriptional enhancement suggests that MARs are important for gene expression and not just for DNA packaging.

Animals↗

PubMed1993

Identification and analysis of a matrix-attachment region 5' of the rat glutamate-dehydrogenase-encoding gene.

Eukaryotic chromatin is thought to be organized into independently regulated loop domains by interaction of matrix-attachment regions (MAR) of the DNA to the nuclear matrix. To define the borders of the chromatin loop containing the glutamate dehydrogenase (GDH) gene, we screened the GDH gene and flanking regions for the presence of MAR sequences. We here report identification, mapping and sequencing of an (A + T)-rich MAR located 2010-1397 bp upstream of the transcription initiation site of GDH, that mediates strong binding to the nuclear matrix. Smaller regions can also confer binding capacity, although at a lower affinity. This (A + T)-rich MAR contained 11 bp and 12 bp (A + T)-rich direct repeats, but not any of the sequences previously described to be associated with MAR activity. We here show that the presence of (A + T)-rich domains of DNA is not sufficient to confer binding capacity, since (A + T)-rich sequences located downstream of the identified MAR did not bind to the nuclear matrix. Moreover, a consensus topoisomerase-II-binding site located downstream of the MAR was found to be insufficient to mediate substantial binding. The number of binding sites in the nuclear matrix for MAR-containing fragments was shown to be approximately 15,000/nucleus. Since organization of the entire rat genome in loops with an average loop size of 100 kbp would require 60,000 binding sites, this suggests that only part of the genome is organized in loops. Alternatively, we might have underestimated the number of binding sites. The GDH MAR, and MAR-containing fragments derived from other species, were found to bind to the same binding sites in the nuclear matrix, although the affinity varied.

Animals↗

PubMed2006

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.

Base Composition↗

PubMed2005

An inactive X specific replication origin associated with a matrix attachment region in the human X linked HPRT gene.

Early in female mammalian embryogenesis, one of the two X chromosomes is inactivated to compensate the gene dosage between males and females. One of the features of X chromosome inactivation (XCI) is the late replication of the inactivated X chromosome. This study reports the identification, by competitive PCR of nascent DNA, of a replication origin in intron 2 of the human X-linked HPRT gene, that is functional only on the inactive X. Features frequently associated with replication origins, including a peak of enhanced DNA flexibility, a perfect match to the yeast ACS sequence, a 14/15 match to the Drosophila topoisomerase II consensus, and a 20/21 match to an initiation region consensus sequence, were identified close to the replication origin. The origin is located approximately 2 kb upstream of a matrix attachment region (MAR) and also contains two A:T-rich elements, thought to facilitate DNA unwinding.

Animals↗

PubMed1995

Nucleolin is a matrix attachment region DNA-binding protein that specifically recognizes a region with high base-unpairing potential.

A DNA affinity column containing a synthetic double-stranded nuclear matrix attachment region (MAR) was used to purify a 100-kDa protein from human erythroleukemia K562 cells. This protein was identified as nucleolin, the key nucleolar protein of dividing cells, which is thought to control rRNA gene transcription and ribosome assembly. Nucleolin is known to bind RNA and single-stranded DNA. We report here that nucleolin is also a MAR-binding protein. It binds double-stranded MARs from different species with high affinity. Nucleolin effectively distinguishes between a double-stranded wild-type synthetic MAR sequence with a high base-unpairing potential and its mutated version that has lost the unpairing capability but is still A+T rich. Thus, nucleolin is not merely an A+T-rich sequence-binding protein but specifically binds the base-unpairing region of MARs. This binding specificity is similar to that of the previously cloned tissue-specific MAR-binding protein SATB1. Unlike SATB1, which binds only double-stranded MARs, nucleolin binds the single-stranded T-rich strand of the synthetic MAR probe approximately 45-fold more efficiently than its complementary A-rich strand, which has an affinity comparable to that of the double-stranded form of the MAR. In contrast to the high selectivity of binding to double-stranded MARs, nucleolin shows only a small but distinct sequence preference for the T-rich strand of the wild-type synthetic MAR over the T-rich strand of its mutated version. The affinity to the T-rich synthetic MAR is severalfold higher than to its corresponding RNA and human telomere DNA. Quantitative cellular fractionation and extraction experiments indicate that nucleolin is present both as a soluble protein and tightly bound to the matrix, similar to other known MAR-binding proteins.

Base Sequence↗

PubMed1991

Nuclear matrix attachment regions and topoisomerase II binding and reaction sites in the vicinity of a chicken DNA replication origin.

We have mapped nuclear matrix attachment regions (MARs), defined by their specific binding to nuclear matrices in vitro, and sites of topoisomerase II reaction, detected by DNA cleavage in vitro in the presence of the inhibitor VM-26, in the vicinity of the replication origin of the chicken alpha-globin gene domain. Two MARs are located close to the downstream end (in the direction of transcription) of a 3 kb fragment which includes the origin. These MARs contain sites for strong topoisomerase II binding and reaction. Our observations on this gene domain support two hypotheses concerning MARs in eukaryotic cells, namely that they are close to DNA replication origins and that they contain multiple topoisomerase II recognition sites.

Animals↗

PubMed1996

A matrix attachment region (MAR)-binding activity due to a p114 kilodalton protein is found only in human breast carcinomas and not in normal and benign breast disease tissues.

A M(r) 114,000 protein (p114) that specifically binds to nuclear matrix attachment DNA (matrix attachment region, MAR) from a breast carcinoma cell line SK-BR-3 was purified to near homogeneity. p114 strongly binds to a wild-type A+T-rich MAR probe with high unwinding propensity with a dissociation constant (Kd) of 10(-9), while it exhibits substantially reduced binding to a mutated A+T-rich non-MAR probe, which lacks unwinding propensity. This binding specificity and affinity is similar to the previously cloned thymocyte-associated MAR-binding protein SATB1. By Southwestern blot analysis, the MAR-binding activity of p114 is detectable in human breast carcinomas but is undetectable in normal breast tissues, benign breast diseases, and immortalized epithelial MCF-10A cells. Thus, the MAR-binding activity of p114 is not merely reflecting cell proliferation, but it strongly associates with breast carcinomas. The p114 MAR-binding activity was found in all 43 human breast carcinoma specimens tested, without exception. Much stronger p114 MAR-binding activity was detected in poorly differentiated than well-differentiated carcinomas. p114 may be a reliable diagnostic and possibly prognostic marker for breast cancer.

Base Sequence↗