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Results for “Matrix Attachment Regions”

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PubMed1998

Matrix attachment region sequences enhanced the expression frequency of a whey acidic protein/human lactoferrin fusion gene in the mammary gland of transgenic mice.

To elevate the expression frequency of transgenes in transgenic mice, the chicken lysozyme matrix attachment region (MAR) sequence was used by combining it with a transgene. The whey acidic protein (WAP) promoter/human lactoferrin (hLF) cDNA fusion transgene (pWL) was connected to the chicken lysozyme MAR sequence at its 5'-end (pMWL). While only two of three mice became transgenic from the pWL vector expressed hLF, all seven mice from the pMWL vector expressed the transgene in their lactating mammary glands. To evaluate the effect of lactogenic hormones on transgene expression, experiments with the primary culture of transgenic mammary explants were performed. It was revealed that the expression of transgenes was slightly increased by insulin plus dexamethasone or insulin plus prolactin treatment. However it was not increased by insulin, dexamethasone or prolactin (IDP) treatment alone. In contrast, the endogenous WAP gene was expressed only in the IDP treated group. These results demonstrate that MAR sequences are effective in improving the expression frequency of transgenes in transgenic mice although the developmental and hormonal regulations are not the same as those of the endogenous WAP gene.

Animals↗

PubMed2001

Small tumor virus genomes are integrated near nuclear matrix attachment regions in transformed cells.

More than 15% of human cancers have a viral etiology. In benign lesions induced by the small DNA tumor viruses, viral genomes are typically maintained extrachromosomally. Malignant progression is often associated with viral integration into host cell chromatin. To study the role of viral integration in tumorigenesis, we analyzed the positions of integrated viral genomes in tumors and tumor cell lines induced by the small oncogenic viruses, including the high-risk human papillomaviruses, hepatitis B virus, simian virus 40, and human T-cell leukemia virus type 1. We show that viral integrations in tumor cells lie near cellular sequences identified as nuclear matrix attachment regions (MARs), while integrations in nonneoplastic cells show no significant correlation with these regions. In mammalian cells, the nuclear matrix functions in gene expression and DNA replication. MARs play varied but poorly understood roles in eukaryotic gene expression. Our results suggest that integrated tumor virus genomes are subject to MAR-mediated transcriptional regulation, providing insight into mechanisms of viral carcinogenesis. Furthermore, the viral oncoproteins serve as invaluable tools for the study of mechanisms controlling cellular growth. Similarly, our demonstration that integrated viral genomes may be subject to MAR-mediated transcriptional effects should facilitate elucidation of fundamental mechanisms regulating eukaryotic gene expression.

Attachment Sites, Microbiological↗

PubMed1995

Mutually exclusive interaction of a novel matrix attachment region binding protein and the NF-muNR enhancer repressor. Implications for regulation of immunoglobulin heavy chain expression.

The immunoglobulin heavy chain (IgH) intronic enhancer stimulates transcription from functional promoters in B lymphocytes but not other cell types. The observation that binding sites for the nuclear factor-mu negative regulator (NF-muNR) enhancer repressor overlap nuclear matrix attachment regions (MARs) in this enhancer has lead to the hypothesis that the cell type specificity of the enhancer might be controlled by regulating nuclear matrix attachment (Scheuermann, R. H., and Chen, U. (1989) Genes & Dev. 3, 1255-1266). To understand the role of MARs in IgH enhancer regulation, we have identified a novel MAR-binding protein, MAR-BP1, from soluble nuclear matrix preparations based on its ability to bind to the MARs associated with the IgH enhancer. Purified MAR-BP1 migrates as a 33-kDa protein, and it can be found in nuclear matrix preparations from a number of different types of lymphoid cell lines. Although specific binding sites have been difficult to localize by chemical or enzymatic footprinting procedures, NF-muNR binding sites are critical for efficient MAR-BP1 binding. Indeed, binding of the IgH enhancer to either intact nuclear matrix preparations or to MAR-BP1 is mutually exclusive to NF-muNR binding. These results are consistent with a model for cell-type specific regulation in which binding of the NF-muNR repressor to the IgH enhancer prevents nuclear matrix attachment in inappropriate cells by interfering with MAR-BP1/enhancer interaction.

Animals↗

PubMed1998

Deletion of the IgH intronic enhancer and associated matrix-attachment regions decreases, but does not abolish, class switching at the mu locus.

The IgH locus intronic enhancer (Emu), located in the intron between the J(H) segments and the Cmu gene, and flanked by two matrix attachment regions (MAR), has been shown to be a major regulator of IgH gene transcription and VDJ recombination. To define the potential role of Emu plus MAR in class switch recombination (CSR), we generated IgG-expressing hybridomas from B cells heterozygous for mutations that delete all of these elements or replace them with a neo(r) gene and analyzed the switch status of the mutated IgH loci. Emu/MAR-deleted IgH loci displayed a highly significant, although not complete, decrease in CSR when compared to unmutated loci in normal hybridomas. Surprisingly, mutant loci with a pgk promoter-driven neo(r) gene replacing the Emu/MAR showed relatively normal switch frequency. These findings indicate that the Emu/MAR region plays a significant, but not necessary role in facilitating class switching at the mu locus. Potential mechanisms for these findings are discussed.

Age Factors↗

PubMed2006

Inclusion of a matrix-attached region in a 7SK pol III vector increases the efficiency of shRNA-mediated gene silencing in embryonic carcinoma cells.

RNA interference is a widely used tool for analysis of gene function in mammalian cells. Stable knockdown of specific target genes can be maintained in cell lines and live organisms using vector-based delivery of short hairpins (shRNAs) driven by RNA polymerase III promoters. Here we describe a vector incorporating the human 7SK promoter for shRNA-mediated gene silencing in the P19 embryonic carcinoma stem cell line. Our preliminary experiments with the 7SK shRNA expression vector indicated that its activity could be hindered by random genomic integration. In order to counter this inhibitory mechanism, we inserted a matrix-attached region sequence to generate an episomal vector system. We compared the effects of insertion versus exclusion of the MAR sequence on the shRNA-mediated gene-specific silencing of the beta-tubulin III and Cyclophilin A genes. While the MAR sequence is not strongly correlated with the episomal status of the expression vector, our studies indicate that inclusion of the MAR element significantly enhances the stability of shRNA-mediated gene silencing in the P19 stem cells.

Base Sequence↗

PubMed2005

Novel transcription factor Satb2 interacts with matrix attachment region DNA elements in a tissue-specific manner and demonstrates cell-type-dependent expression in the developing mouse CNS.

Satb1 is a first cell-type-specific transcription factor of a novel type that functions as a regulator of the transcription of large chromatin domains. We identified a close homologue of Satb1, Satb2, in a cDNA subtraction screening in a search for genes controlling neural differentiation. Satb2 showed 61% amino-acid homology to Satb1. Satb2 and Satb1 expression was detected in different cell subpopulations of developing mouse CNS in a mutually exclusive manner. In the electrophoretic mobility shift assay we demonstrate that nuclear extracts from the embryonic day 18.5 mouse developing neocortex, in contrast to basal ganglia, contain a protein complex interacting with matrix attachment region DNA elements (MARs) with high affinity. Endogenous Satb2 protein is a part of this complex. In the developing neocortex Satb2 was detected largely in the superficial layers. In the developing spinal cord Satb2 expression marks a subpopulation of Lbx1-positive neurons dorsally and a subgroup of Isl1-positive neurons ventrally. In the Lbx1 mutants Satb2 expression is greatly reduced. We suggest that Satb2 may regulate differentiation of subsets of neurons at the level of higher order chromatin structure via binding to MARs.

Amino Acid Sequence↗

PubMed1996

Co-integration of beta-lactoglobulin/human serum albumin hybrid genes with the entire beta-lactoglobulin gene or the matrix attachment region element: repression of human serum albumin and beta-lactoglobulin expression in the mammary gland and dual regulation of the transgenes.

The effect of co-integration of the entire beta-lactoglobulin (BLG) gene or matrix attachment region (MAR) sequences on the expression of various BLG/ human serum albumin (HSA) gene constructs was tested in transgenic mice. These former sequences were chosen because of their reported ability to insulate transgenes from the neighboring host genomic DNA sequences and/or to provide a more permissive transcriptional environment. When introduced alone, a cDNA-based BLG/HSA construct was expressed in 60% of transgenic strains and HSA was secreted at levels up to 0.3 mg/ml into the milk. Upon co-integration with either the entire BLG gene or MAR element, HSA RNA and protein expression were completely abrogated. While the co-integrated BLG gene suppressed the proportion of expresser strains carrying cDNA as well as genomic BLG/HSA constructs, the MAR element only exerted its negative effect on the cDNA-based BLG/HSA construct. In transgenics expressing both HSA and BLG, the tissue specificity and developmental patterns of BLG expression were altered and resembled the less stringent pattern of the BLG/HSA expression. These results demonstrate that rescue of transgene expression through co-integration with BLG or MAR sequences do not apply universally.

Animals↗

PubMed2004

Stable high-level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions.

Basic and applied research involving transgenic plants often requires consistent high-level expression of transgenes. However, high inter-transformant variability of transgene expression caused by various phenomena, including gene silencing, is frequently observed. Here, we show that stable, high-level transgene expression is obtained using Arabidopsis thaliana post-transcriptional gene silencing (PTGS) sgs2 and sgs3 mutants. In populations of first generation (T1) A. thaliana plants transformed with a beta-glucuronidase (GUS) gene (uidA) driven by the 35S cauliflower mosaic virus promoter (p35S), the incidence of highly expressing transformants shifted from 20% in wild type background to 100% in sgs2 and sgs3 backgrounds. Likewise, when sgs2 mutants were transformed with a cyclin-dependent kinase inhibitor 6 gene under control of p35S, all transformants showed a clear phenotype typified by serrated leaves, whereas such phenotype was only observed in about one of five wild type transformants. p35S-driven uidA expression remained high and steady in T2 sgs2 and sgs3 transformants, in marked contrast to the variable expression patterns observed in wild type T2 populations. We further show that T-DNA constructs flanked by matrix attachment regions of the chicken lysozyme gene (chiMARs) cause a boost in GUS activity by fivefold in sgs2 and 12-fold in sgs3 plants, reaching up to 10% of the total soluble proteins, whereas no such boost is observed in the wild type background. MAR-based plant transformation vectors used in a PTGS mutant background might be of high value for efficient high-throughput screening of transgene-based phenotypes as well as for obtaining extremely high transgene expression in plants.

Arabidopsis↗

PubMed2000

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.

Alcohol Dehydrogenase↗

PubMed1997

Extension of chromatin accessibility by nuclear matrix attachment regions.

Transcription of the variable region of the rearranged immunoglobulin mu gene is dependent on an enhancer sequence situated within one of the introns of the gene. Experiments with transgenic mice have shown that activation of the promoter controlling this transcription also requires the matrix-attachment regions (MARs) that flank the intronic enhancer. As this mu gene enhancer can establish local areas of accessible chromatin, we investigated whether the MARs can extend accessibility to more distal positions. We eliminated interactions between enhancer- and promoter-bound factors by linking mu enhancer/MAR fragments to the binding sites for bacteriophage RNA polymerases that were either close to or one kilobase distal to the enhancer. The mu enhancer alone mediated chromatin accessibility at the proximal site but required a flanking MAR to confer accessibility upon the distal promoter. This long-range accessibility correlates with extended demethylation of the gene construct but not with whether it is being actively transcribed. MARs thus collaborate with the mu enhancer to generate an extended domain of accessible chromatin.

Animals↗

PubMed1992

A transcription-dependent DNase I-hypersensitive site in a far upstream segment of the chicken alpha-globin gene domain coincides with a matrix attachment region.

A site of hypersensitivity to DNase I has been found at the 5'-side of the chicken alpha-globin gene domain, ca. 8 Kbp upstream to the first gene. The presence of this site in different types of chicken cells correlates with the transcriptional status of the domain. A matrix attachment region (MAR) has also been found in the same subfragment, suggesting that it may be involved in the control of transcription of the chicken alpha-globin genes.

Alpharetrovirus↗

PubMed2005

In silico and wet-bench identification of nuclear matrix attachment regions.

Chromatin loops are tethered at discrete regions that are approx 100-1000 bp in length. These regions of attachment serve as specific sequence landmarks, anchoring the DNA to the fibers of the chromosomal scaffold. It has been estimated that our genome contains 70,000 nuclear matrix attachment sites that serve as a dynamic nuclear organizer in both the interphase and metaphase cell. Approximately 30,000-40,000 matrix attachment regions (MARs) serve as origins of replication. MARs can also be associated with chromosomal segments densely populated with transcription factor-binding sites. This may facilitate transcription that is initiated within the region of the chromosome coincident with the surface of the nuclear matrix. Assuming an average somatic loop size of 100 kb, it is reasonable to propose that each cell utilizes 30,000 MARs to anchor each of the approx 20,000 active genic domains. This is sufficient to encompass the 30,000 functional genes in our genome that exist as members of single or multigenic families, each constituting a single chromatin domain. With the sequencing phase of various genome projects complete, in silico tools are being developed to identify the long-range control elements that modulate gene expression. This information is necessary to specifically target the time-intensive wet-bench verification and expression experiments that will provide a unified understanding of gene regulation. In this chapter we review some of the in silico strategies that are currently available and a new in vivo method based on the real-time polymerase chain reaction, to assess regions of matrix association.

Actins↗

PubMed2003

Matrix attachment region (MAR) properties and abnormal expansion of AT island minisatellites in FRA16B fragile sites in leukemic CEM cells.

AT-rich minisatellites (AT islands) are sites of genomic instability in cancer cells and targets for extremely lethal AT-specific drugs, such as bizelesin. Here we investigated the AT islands in the FRA16B fragile site region for their possible roles in the organization of DNA on the nuclear matrix. The FRA16B AT island nominally spans approximately 3 kb of mostly >90% A/T DNA. In silico analysis indicates that this domain exhibits characteristics of nuclear matrix attachment regions (MARs): an exceptionally intense computed 'MAR potential' and profound duplex destabilization and flexibility. FRA16B repeats specifically bind to isolated nuclear matrices, which indicates their in vitro MAR function. This binding is several-fold greater than that of a known MAR in the c-myc gene. AT islands in fragile sites FRA16B and FRA16D are significantly more abundant in CEM cells that are hypersensitive to bizelesin compared to normal WI-38 cells. FRA16B overabundance in CEM is due to an approximately 10-fold expansion of FRA16B repeats. The expanded FRA16B minisatellites in CEM cells preferentially localize to the nuclear matrix-associated DNA indicating their in vivo MAR function. The unexpanded repeats in WI-38 cells localize to the loop DNA. The c-myc MAR is also matrix-associated in CEM cells while localizing to loop DNA in WI-38 cells. These results are the first to demonstrate that AT islands in fragile sites can function as MARs both in vitro and in vivo. The ability of FRA16B-mediated MAR sites to rearrange depending on the repeat expansion status could be relevant to both genomic instability of cancer cells and their sensitivity to AT-island targeting drugs.

AT Rich Sequence↗

PubMedDate not supplied

The effect of matrix attached regions (MAR) and specialized chromatin structure (SCS) on the expression of gene constructs in cultured cells and in transgenic mice.

The flanking sequences of several genes have been shown to direct a position independent expression of transgenes. Attempts to completely identify the insulating sequences have failed so far. Some of these sequences contain a matrix attached region (MAR) located in the flanking part of the genes. This article will show that the MARs in cultured cells located in the 3' OH region of the human apolipoprotein B100 (Apo B100) and within the SV40 genome were unable to stimulate and insultate transgene expression directed by the promoters from a rabbit whey acidic protein (WAP) gene or from human cytomegalovirus (hCMV) early genes. In transgenic mice, the MAR from the Apo B100 and SV40 genes did not enhance the expression of a transgene containing the rabbit whey acid protein (WAP) promotor, the late gene SV40 intron (VP1 intron), the bovine growth hormone (bGH) cDNA and the SV40 late gene terminator. This construct was even toxic for embryos. Similarly, the specialized chromatin structure (SCS) from the Drosophila 87A7 HSP70 gene reduced chloramphenicol acetyl transferase (CAT) activity when added between a cytomegalovirus (CMV) enhancer and a Herpes simplex thymidine kinase (TK) gene promoter. This inhibitory action was almost complete when a second SCS sequence was added before the CMV enhancer. Sequences from the firefly luciferase and from the human gene cathepsin D cDNA used as control unexpectedly showed a similar inhibitory effect when added to the CMVTKCAT construct instead of SCS. When added before the CMV enhancer and after the transcription terminator in the CMVTKCAT construct, the SCS sequence was unable to insulate the integrated gene as seen by the fact that the level of CAT in cell extracts were by no means correlated with the number of copies in individual clones. From these data, it is concluded that i) a MAR containing the canonical AT rich sequences does not amplify the expression of all gene constructs ii) At rich MAR sequences do not have per se an insulating effect iii) Drosophila SCS from the 87A7 HSP70 gene has no insulating effect in all gene constructs (at least in mammalian cells) iv) and the addition of a DNA fragment between an enhancer and a promoter in a gene construct cannot be used as a reliable test to evaluate its insulating property.

Animals↗

PubMed2004

[The potential role of nuclear matrix attachment regions (MARs) in regulation of gene expression].

Gene transfer technology is being used to enhance agronomic performance or improve quality traits in a wide variety of crop species. However, it is sometimes severely handicapped by difficulty in obtaining material in which transgene expression is predictable and stable over many generations. Because integration seemed to occur randomly in the plant genome, it was thought that some transgenes would be integrated in a relatively uncondensed, transcriptionally active chromatin environment, while others in a condensed, transcriptionally inert chromatin structure. Nuclear matrix attachment regions (MARs) are defined as DNA sequences that bind preferentially to the proteins of the nuclear matrix. They typically are localized at the borders of gene domains, implicating them in the formation of individual loops of higher order chromatin structure and transcription regulation. When MARs are positioned on either side of a transgene their presence usually results in higher and more stable espression in transgenic plants, most likely by minimizing gene silencing. In this review, we focus mainly on novel findings and our observations concerning the function of MARs in transcription regulation. Our objective is not only to summarize the current data and present several possible models to explain MAR effects on the transcription regulation, but also to point out some open questions involving the utilization of MARs in constructing high efficient expression vectors.

Chromatin↗

PubMed2003

Stimulation of Tat-independent transcriptional processivity from the HIV-1 LTR promoter by matrix attachment regions.

The chromatin environment and the sites of integration in the host genome are critical determinants of human immunodeficiency virus (HIV) transcription and replication. Depending on the chromosomal location of provirus integration within the genome, HIV-1 long terminal repeat (LTR)-mediated transcription may vary from 0- to 70-fold. Cis-elements such as topoisomerase II cleavage sites, Alu repeats and matrix attachment regions (MARs) are thought to be targets for retroviral integration. Here we show that a novel MAR sequence from the T-cell receptor beta locus (MARbeta) and the IgH MAR mediate transcriptional augmentation when placed upstream of the HIV-1 LTR promoter. The effect of transcriptional augmentation is seen in both transient and stable transfection, indicating its effect even upon integration in the genome. MAR-mediated transcriptional elevation is independent of Tat, and occurs synergistically in the presence of Tat. Further, we show that MAR-mediated transcriptional elevation is specific to the HIV-1 LTR and the Moloney murine leukemia virus LTR promoter. In a transient transfection assay using over-expressed IkappaB, the inhibitor of NF-kappaB, we show that MAR-induced processive transcription is NF-kappaB dependent, signifying the role of local enhancers within the LTR promoter. Furthermore, by RNase protection experiments using proximal and distal probes, we show that MAR-mediated transcriptional upregulation is more prominent at the distal rather than the proximal end, thus indicating the potential role of MARs in promoting elongation.

Animals↗

PubMed2003

Stable transformation of Theobroma cacao L. and influence of matrix attachment regions on GFP expression.

We describe a protocol for Agrobacterium-mediated genetic transformation of Theobroma cacao L. using cotyledonary explants from primary somatic embryos (SEs) and A. tumefaciens strain AGL1. Transgenic plants carrying the visible marker, gene green fluorescent protein ( EGFP), the selectable marker gene neomycin phosphotransferase II ( NPTII), the class I chitinase gene from cacao ( Chi), and tobacco nuclear matrix attachment regions (MARs) in different combinations were successfully produced via regeneration of secondary SEs. The presence of the Chi gene or MARs did not influence the number of transgenic plants produced compared to the marker genes alone. However, the inclusion of MARs contributed to increased mean GFP expression in the population of transgenics. Additionally, the presence of MARs reduced the occurrence of gene silencing and stabilized high levels of GFP expression in lines of transgenic plants multiplied via reiterative somatic embryogenesis. Ninety-four transgenic plants were acclimated in a greenhouse and grown to maturity. Detailed growth analysis indicated that there were no differences in various growth parameters between transgenic and non-transgenic SE-derived plants. Seeds produced from two genetic crosses with one of the transgenic lines were analyzed for EGFP expression-a near-perfect 1:1 segregation was observed, indicating that this line resulted from the insertion of a single locus of T-DNA.

Base Sequence↗

PubMed1998

Nuclear matrix attachment regions of human papillomavirus type 16 point toward conservation of these genomic elements in all genital papillomaviruses.

The gene functions, transcriptional regulation, and genome replication of human papillomaviruses (HPVs) have been extensively studied. Thus far, however, there has been little research on the organization of HPV genomes in the nuclei of infected cells. As a first step to understand how chromatin and suprachromatin structures may modulate the life cycles of these viruses, we have identified and mapped interactions of HPV DNAs with the nuclear matrix. The endogenous genomes of HPV type 16 (HPV-16) which are present in SiHa, HPKI, and HPKII cells, adhere in vivo to the nuclear matrixes of these cell lines. A tight association with the nuclear matrix in vivo may be common to all genital HPV types, as the genomes of HPV-11, HPV-16, HPV-18, and HPV-33 showed high affinity in vitro to preparations of the nuclear matrix of C33A cells, as did the well-known nuclear matrix attachment region (MAR) of the cellular beta interferon gene. Affinity to the nuclear matrix is not evenly spread over the HPV-16 genome. Five genomic segments have strong MAR properties, while the other parts of the genome have low or no affinity. Some of the five MARs correlate with known cis-responsive elements: a strong MAR lies in the 5' segment of the long control region (LCR), and another one lies in the E6 gene, flanking the HPV enhancer, the replication origin, and the E6 promoter. The strongest MAR coincides with the E5 gene and the early-late intergenic region. Weak MAR activity is present in the E1 and E2 genes and in the 3' part of L2. The in vitro map of MAR activity appears to reflect MAR properties in vivo, as we found for two selected fragments with and without MAR activity. As is typical for many MARs, the two segments with highest affinity, namely, the 5' LCR and the early-late intergenic region, have an extraordinarily high A-T content (up to 85%). It is likely that these MARs have specific functions in the viral life cycle, as MARs predicted by nucleotide sequence analysis, patterns of A-T content, transcription factor YY1 binding sites, and likely topoisomerase II cleavage sites are conserved in similar positions throughout all genital HPVs.

Adenine↗