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Results for “Matrix Attachment Regions”

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PubMed1998

A nuclear matrix attachment region upstream of the T cell receptor beta gene enhancer binds Cux/CDP and SATB1 and modulates enhancer-dependent reporter gene expression but not endogenous gene expression.

We have previously identified a DNase I-hypersensitive site in the T cell receptor beta locus, designated HS1, that is located 400 base pairs upstream of the transcriptional enhancer Ebeta and is induced during CD4(-)CD8(-) to CD4(+)CD8(+) thymocyte differentiation. Using electrophoretic mobility shift assays, we show that HS1 induction correlates with increased binding of two nuclear factors, Cux/CDP and SATB1, to a 170-base pair DNA sequence within HS1. Furthermore, we demonstrate that HS1 is a nuclear matrix attachment region, referred to as MARbeta. These findings demonstrate that an analogous organization of cis-regulatory elements in which a nuclear matrix attachment region is in close proximity to an enhancer is conserved in the immunoglobulin and T cell receptor loci. In addition, we show that MARbeta represses Ebeta-dependent reporter gene expression in transient transfection assays. However, the targeted deletion of MARbeta from the endogenous locus does not change T cell receptor beta gene transcription in developing T cells. These contrasting results suggest a potential pitfall of functional studies of nuclear matrix attachment regions outside of their natural chromosomal context.

Animals↗

PubMed1999

MAF1, a novel plant protein interacting with matrix attachment region binding protein MFP1, is located at the nuclear envelope.

The interaction of chromatin with the nuclear matrix via matrix attachment region (MAR) DNA is considered to be of fundamental importance for chromatin organization in all eukaryotic cells. MAR binding filament-like protein 1 (MFP1) from tomato is a novel plant protein that specifically binds to MAR DNA. Its filament protein-like structure makes it a likely candidate for a structural component of the nuclear matrix. MFP1 is located at nuclear matrix-associated, specklelike structures at the nuclear envelope. Here, we report the identification of a novel protein that specifically interacts with MFP1 in yeast two-hybrid and in vitro binding assays. MFP1 associated factor 1 (MAF1) is a small, soluble, serine/threonine-rich protein that is ubiquitously expressed and has no similarity to known proteins. MAF1, like MFP1, is located at the nuclear periphery and is a component of the nuclear matrix. These data suggest that MFP1 and MAF1 are in vivo interaction partners and that both proteins are components of a nuclear substructure, previously undescribed in plants, that connects the nuclear envelope and the internal nuclear matrix.

Amino Acid Sequence↗

PubMed1997

Matrix-attachment regions in the mouse chromosome 7F imprinted domain.

We have mapped the matrix-attachment regions (MARs) in 200 kilobases of the mouse Chromosome (Chr) 7F imprinted domain. MARs are genetic elements known to have effects in cis on methylation at nonimprinted loci. The imprinting of the Igf2 and Ins2 genes is dependent on the transcription of the downstream H19 gene. The transcription of H19 is dependent in turn on its methylation status. The cis-acting regulators of methylation at this site are not known. As MARs are potential regulators not only of methylation but also other elements of genomic imprinting, we mapped the MARs within the 200 kilobases around H19. This report describes the mapping of four MARs from this region.

Animals↗

PubMed2004

Human matrix attachment regions are necessary for the establishment but not the maintenance of transgene insulation in Drosophila melanogaster.

Human matrix attachment regions (MARs) can insulate transgene expression from chromosomal position effects in Drosophila melanogaster. To gain insight into the mechanism(s) by which chromosomal insulation occurs, we studied the expression phenotypes of Drosophila transformants expressing mini-white transgenes in which MAR sequences from the human apoB gene were arranged in a variety of ways. In agreement with previous reports, we found that a single copy of the insulating element was not sufficient for position-independent transgene expression; rather, two copies were required. However, the arrangement of the two elements within the transgene was unimportant, since chromosomal insulation was equally apparent when both copies of the insulator were upstream of the mini-white reporter as when the transcription unit was flanked by insulator elements. Moreover, experiments in which apoB 3' MAR sequences were removed from integrated transgenes in vivo by site-specific recombination demonstrated that MAR sequences were required for the establishment but not for the maintenance of chromosomal insulation. These observations are not compatible with the chromosomal loop model in its simplest form. Alternate mechanisms for MAR function in this system are proposed.

Animals↗

PubMed2005

TM2, a novel strong matrix attachment region isolated from tobacco, increases transgene expression in transgenic rice calli and plants.

Nuclear matrix attachment regions (MARs) are thought to influence the expression of the flanking genes. TM2, a new DNA fragment isolated from tobacco, can bind with the rice nuclear matrix in vitro. In this study, we investigated the effect of TM2 on transgene expression under the control of three different promoters in stably transformed rice calli and plants. The presence of TM2 flanking the transgene increased the expression of constructs based on the constitutive CaMV 35S and maize ubiquitin gene promoters in both resistant calli and transformed plants. The GUS expression directed by the photosynthetic-tissue-specific PNZIP promoter was also increased in photosynthetic tissues of transformants. However, TM2 did not change the gene expression pattern controlled by the PNZIP promoter. The effect of TM2 in transgenic plants was stronger than that in transgenic calli based on all three promoters. Our results indicate that TM2, as a novel strong MAR, can be used to increase the transgene expression levels in the whole plant or in particular tissues of monocotyledons.

Base Sequence↗

PubMed2026

A CHO-Derived Matrix Attachment Region Enhances Transgene Dosage, SATB1 Recruitment, and Monoclonal Antibody Expression in a Dual-Promoter Vector System.

The production of monoclonal antibodies (mAbs) in Chinese hamster ovary (CHO) cells is often affected by position-effect variegation and the gradual loss of transgene expression over time. Hence, we have designed a dual-promoter IgG expression vector and compared versions that either contained or lacked a CHO-derived matrix-attachment region (MAR). Stable CHO-S pools, cultured in serum-free conditions, revealed that the MAR-containing construct produced higher and more consistent antibody levels across ten passages, as confirmed by Western blot and Protein A Octet analysis. Product-quality analysis by size-exclusion chromatography and reducing SDS-PAGE confirmed formation of properly assembled, mainly monomeric antibodies in both cases. Quantitative PCR indicated greater transgene copy numbers in MAR pools (+ 48% for the light chain and + 71% for the heavy chain), and RT-qPCR showed roughly fourfold higher transcript levels for both chains relative to controls. Bioinformatic analysis revealed several SATB1 binding motifs within the MAR sequence, and ChIP-qPCR demonstrated SATB1 association with the MAR-linked transgene locus. Overall, the data suggested that a CHO-native MAR could enhance transgene dosage and transcriptional activity, while preserving product integrity, possibly through SATB1-mediated chromatin organization. Ongoing work includes chromatin-mark profiling and process-level productivity measurements to better define the impact of MAR-based vector design on biomanufacturing performance.

Animals↗

PubMed2005

Hypothesis: biological role for J-C intronic matrix attachment regions in the molecular mechanism of antigen-driven somatic hypermutation.

A major function of J-C intronic matrix attachment regions (MAR) during immune diversification via somatic hypermutation (SHM) at immunoglobulin loci may be to manipulate the topology of DNA within the upstream target domain. The suggestion that SHM induction requires MAR-induced torsional strain, in conjunction with DNA remodelling at the J-C intron, completes the definition of a cogent paradigm within which all extant molecular data on the issue may be interpreted. Moreover, the suggestion that a mutagenic mechanism relieves MAR-generated superhelicity could provide an indication as to the evolutionary basis of SHM.

Antigens↗

PubMed2002

Matrix attachment regions (MARs) enhance transformation frequencies and reduce variance of transgene expression in barley.

Nuclear matrix attachment regions (MARs) are defined as genomic DNA sequences, located at the physical boundaries of chromatin loops. They are suggested to play a role in the cis unfolding and folding of the chromatin fibre associated with the regulation of gene transcription. Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. The present study is the first to investigate the influence of MAR sequences on transformation frequencies and transgene expression in barley, which is highly relevant to the future improvement of this crop by biotechnology. Two plant MAR sequences were tested both for their ability to bind to the nuclear matrix of barley leaf nuclei and to regulate the expression of a reporter gene in transgenic barley. Competitive in vitro MAR binding assays with the 520 bp P1-MAR from soybean and the 516 bp TBS-MAR from petunia revealed that only the P1-MAR had specific binding affinity for barley nuclear matrices. The barley transformation frequency with the uidA reporter gene was increased 2-fold when the gene was flanked with either the P1-MAR or TBS-MAR, while the gene copy number was strongly reduced. The presence of P1-MAR sequences increased the mean activity and reduced the variance in expression of a co-integrated reporter gene in barley consistent with the proposed model of MAR activity.

Cells, Cultured↗

PubMed2005

Matrix attachment regions increase the efficiency and stability of RNA-mediated resistance to tomato spotted wilt virus in transgenic tobacco.

Matrix attachment regions (MARs) are DNA elements that can increase and stabilize transgene expression. We investigated the effect of the RB7 MAR on transgenic virus resistance. Constructs for resistance to tomato spotted wilt virus (TSWV) with and without flanking RB7 MARs were used to transform tobacco and produce homozygous lines. The population with the MAR construct had a significantly higher percentage of TSWV resistant plants in the R1 generation than the nonMAR population. Each resistant line was advanced to the R4 generation, and significantly fewer MAR lines lost resistance over generations compared to the nonMAR population. Lines with TSWV resistance in growth chamber tests were also resistant in field trials. Two lines that were resistant in the R1 generation and susceptible in the R4 were examined in more detail in order to determine if transcriptional silencing of the transgene was occurring in the later generation. Short interfering 21-25 nt RNAs from the transgene that are characteristic of post-transcriptional gene silencing (PTGS) were present in the resistant R1 plants, but not the susceptible R4 plants, indicating that virus resistance was associated with PTGS of the transgene. Loss of resistance was accompanied by an increase in promoter methylation in both lines. In line M41, the transgene was fully silenced at the transcriptional level in the R4 as shown by nuclear run-on assays. In line NM13, transgene transcription and RNA accumulation was still present in the R4 generation, but the level of transcription was not sufficient to trigger PTGS, suggesting that this line may have partial transcriptional silencing. These results are consistent with the concept that MARs may prevent transcriptional silencing.

Breeding↗

PubMed1994

Characterisation of two intronic nuclear-matrix-attachment regions in the human DNA topoisomerase I gene.

We identify two high-affinity matrix-attachment regions (MAR elements) located in two introns of the human DNA topoisomerase I gene (TOP1). These intronic MAR elements, designated MI and MII, are specifically bound by the nuclear matrix and partition with scaffolds in vitro. One of these MAR elements, MII, is part of a genomic region which is hypersensitive for endogenous nucleases. We have sequenced both DNA elements and have characterized their mode of binding to the nuclear matrix. Experiments with the minor-groove-binding ligands distamycin and chromomycin indicate that the A+T-rich regions, most likely homopolymeric A tracts, are responsible for binding of these DNA elements to the nuclear matrix. MII contains an alu-like element and a segment of curved DNA. Analysis of subfragments of MII show that the curved DNA region itself shows only weak nuclear-matrix binding, and that the high-affinity binding sites are located on subfragments on the 5' side of the curved DNA. In addition, we found that the alu-like sequence does not contribute significantly to the binding of MII and of subfragments of MII to nuclear-matrix proteins. Comparing the distribution of repetitive sequences in the cloned parts of human DNA topoisomerase I gene with the location of high-affinity matrix-binding sites we find no evidence that repetitive DNA may be located close to MAR elements as has been previously suggested.

Binding Sites↗

PubMed2003

Multiple cis-acting sequences implicate function diversity in nuclear matrix attachment regions of bovine mammary gland.

Chromosomal DNA in higher eukaryotes is spatially organized into loops by periodic attachment to the nuclear matrix at its base via a specific matrix attachment region (MAR). In order to study the nature of DNA sequences that affixed the loops to the nuclear matrix, we have cloned the MAR DNA from bovine lactating mammary tissues. In vitro binding assay showed that the cloned fragments could be co-complexed with nuclear matrix proteins to form insoluble complex easily removed by centrifugation. Sequences of the two chosen MAR loci are composed of TG-, CA- and GA- blocks, as well as the ATTA motifs. Both the MAR loci show numerous replication/transcription factor binding sites, enhancer motifs, several perfect or imperfect inverted repeats, and sequences sharing the common features of the potential DNA bending core sequence. The possibility that a combination of different elements in the same DNA sequence may function as either positive or negative regulatory elements in controlling a variety of cellular and developmental processes is discussed.

Animals↗

PubMed2001

Development of stable cell lines for production or regulated expression using matrix attachment regions.

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection procedure. Given the variation in the expression levels of the same construct in individual clones, hundreds of clones must be isolated and tested to identify one or more with the desired characteristics. Various boundary elements (BEs), matrix attachment regions, and locus control regions (LCRs) were screened for their ability to augment the expression of heterologous genes in Chinese hamster ovary (CHO) cells. Of the chromatin elements assayed, the chicken lysozyme matrix-attachment region (MAR) was the only element to significantly increase stable reporter expression. We found that the use of the MAR increases the proportion of high-producing clones, thus reducing the number of clones that need to be screened. These benefits are observed both for constructs with MARs flanking the transgene expression cassette, as well as when constructs are co-transfected with the MAR on a separate plasmid. Moreover, the MAR was co-transfected with a multicomponent regulatable beta-galactosidase expression system in C2C12 cells and several clones exhibiting regulated expression were identified. Hence, MARs are useful in the development of stable cell lines for production or regulated expression.

Animals↗

PubMed2001

Plasmids with a mammalian replication origin and a matrix attachment region initiate the event similar to gene amplification.

Gene amplification plays a crucial role in the development of many human malignancies. Amplified genes are frequently localized on double minutes (DMs). We show here that plasmids bearing both a mammalian replication origin and a nuclear matrix attachment region were able to integrate into DMs if transfected to cells having DMs (COLO 320DM). Furthermore, these plasmids triggered the events leading to the de novo formation of the structure similar to DMs if transfected to the cells without DMs (COLO 320HSR or HeLa). Autonomous replication of these plasmids was suggested to be a prerequisite for these events to occur, because the presence of the origin sequences in the plasmids was required. The presence of matrix attachment region in the plasmids is also required for these events to occur, suggesting that matrix attachment plays an indispensable role in extrachromosomal replication. This model system will allow us to investigate the mechanism of gene amplification as well as to analyze the autonomous replication of the plasmid with mammalian replication origins.

Colorectal Neoplasms↗

PubMed2003

A significant fraction of conserved noncoding DNA in human and mouse consists of predicted matrix attachment regions.

Noncoding DNA in the human-mouse orthologous intergenic regions contains "islands" of conserved sequences, the functions of which remain largely unknown. We hypothesized that some of these regions might be matrix-scaffold attachment regions, MARs (or S/MARs). MARs comprise one of the few classes of eukaryotic noncoding DNA with an experimentally characterized function, being involved in the attachment of chromatin to the nuclear matrix, chromatin remodeling and transcription regulation. To test our hypothesis, we analyzed the co-occurrence of predicted MARs with highly conserved noncoding DNA regions in human-mouse genomic alignments. We found that 11% of the conserved noncoding DNA consists of predicted MARs. Conversely, more than half of the predicted MARs co-occur with one or more independently identified conserved sequence blocks. An excess of conserved predicted MARs is seen in intergenic regions preceding 5' ends of genes, suggesting that these MARs are primarily involved in transcriptional control.

Animals↗

PubMed2000

A matrix attachment region is located upstream from the high-molecular-weight glutenin gene Bx7 in wheat (Triticum aestivum L.).

A 2.2-kb nucleotide sequence rich in AT, located upstream from the Bx7 allele of the high-molecular-weight glutenin Glu-B1 locus in wheat (Triticum aestivum cv. Glenlea) was cloned following amplification by PCR. The 5' region of this sequence contains motifs typically found in matrix attachment regions (MARs) in other plants. We have shown that part of the 2.2-kb DNA binds to wheat nuclear matrix (NM) in vitro, at least as strongly as a known MAR (Adh1) from maize suggesting that there is a MAR upstream of Bx7. This MAR is approximately 800 bases in length running from -750 to -1560 bases, relative to the start codon. Although the MAR is associated with a tissue-specific gene and is beside a strong tissue-specific promoter, the MAR sequence did not lead to tissue-specific expression of the beta-glucuronidase marker gene under the control of the rice actin promoter in various tissues. Presence of the MAR was only slightly beneficial with respect to expression levels, which were not greatly altered in transient expression assays in various wheat tissues although a slight increase in the number of foci was observed in leaves, which have low transformation efficiencies.

Chromatin↗

PubMed1992

A nuclear matrix attachment region organizes the Epstein-Barr viral plasmid in Raji cells into a single DNA domain.

The extrachromosomal Epstein-Barr virus (EBV) plasmid in the Burkitt lymphoma cell line, Raji, is stably associated with the nuclear matrix. This association is effected by a nuclear matrix attachment region (MAR) located in the BamHI C fragment of the viral genome; no other region of EBV DNA was found to be attached to the nuclear matrix with high affinity. The MAR was mapped to 5.2 kbp of DNA, greater than 80% of which is found on the nuclear matrix in unsynchronized cells expressing only viral latent cycle products. Thus the majority of viral plasmids in Raji cells use the same MAR. The MAR of EBV DNA contains the origin of latent viral DNA replication (oriP), the genes for the small viral RNAs (EBERs) and a 500 bp region immediately upstream of the EBER-1 gene. The clustering of the latent viral replication origin and the nearby enhancer and promoters for latent viral transcription on the nuclear matrix is likely to be crucial for regulation of the latent viral genome.

Burkitt Lymphoma↗

PubMed2001

The matrix attachment regions (MARs) associated with the Heat Shock Cognate 80 gene ( HSC80) of tomato represent specific regulatory elements.

Matrix Attachment Regions (MARs) flank certain plant genes and appear in certain cases to be necessary for their proper regulation. For example, we previously demonstrated that the MARs and introns from the Heat Shock Cognate 80 gene of tomato (HSC80) are necessary for efficient expression of HSC80-based transgenes. MARs may exert their effect by anchoring the ends of a chromatin loop to the nuclear matrix, thereby establishing an independent chromatin domain. Alternatively, MARs may facilitate interactions between activating complexes and DNA. In the first case, MARs should enhance the expression of most genes, while in the latter case, their action might be gene-specific. We addressed this problem by testing whether the HSC80 MARs affected the regulation of an unrelated transgene. We constructed a chimeric transgene composed of the Arabidopsis ADENINE PHOSPHORIBOSYLTRANSFERASE (APT) promoter fused to the maize gene Lc, which encodes a regulator of anthocyanin synthesis, and compared the expression of Lc in Arabidopsis transparent testa glabra (ttg) mutants (which lack anthocyanin pigments) transformed with transgene constructs incorporating the MARs or control DNA fragments that do not bind to the nuclear matrix. Quantitative RT-PCR analysis was used to compare Lc expression in the different transgenic lines. Whether the APT-Lc transgene was flanked by the HSC80 MARs or a control fragment had no effect on expression, while the use of a different MAR, the ARS1 MAR from yeast, significantly decreased expression (P=0.03). Comparison of single-copy and multicopy T-DNA insertions indicated that neither the HSC80 MARs nor the ARS1 MAR could protect the APT-Lc transgene from the negative effect of the integration of multiple copies. In conclusion, this work supports a model in which different regulatory elements within the HSC80 locus interact with the nuclear matrix to induce transcriptional competence.

Adenine Phosphoribosyltransferase↗

PubMed1992

Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice.

Matrix-attachment regions (MARs) may function as domain boundaries and partition chromosomes into independently regulated units. We have tested whether MAR sequences from the chicken lysozyme locus, the so-called A-elements, can confer position-independent regulation to a whey acidic protein (WAP) transgene in mammary tissue of mice. In the absence of MARs, expression of WAP transgenes was observed in 50% of the lines, and regulation during pregnancy, during lactation, and upon hormonal induction did not mimic that of the endogenous WAP gene and varied with the integration site. In contrast, all 11 lines in which WAP transgenes were juxtaposed to MAR elements showed expression. Accurate position-independent hormonal and developmental regulation was seen in four out of the five lines analyzed. These results indicate that MARs can establish independent genetic domains in transgenic mice.

Animals↗