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Results for “Matrix Attachment Regions”

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At least 181 records · Page 10Linked to original sources

PubMed1991

Precise localization of the alpha-globin gene cluster within one of the 20- to 300-kilobase DNA fragments released by cleavage of chicken chromosomal DNA at topoisomerase II sites in vivo: evidence that the fragments are DNA loops or domains.

We have mapped the position of the alpha-globin gene cluster in the 20- to 300-kilobase fragments of chromosomal DNA isolated from growing chicken HD3 erythroblastoid cells exposed to 4'-demethylepipodophyllotoxinthenylidene beta-D-glucoside. This epipodophyllotoxin traps functioning topoisomerase II molecules, the denaturation of which cleaves DNA and reveals their reaction sites. The DNA fragments, prepared by centrifugation in sucrose gradients, bind selectively to glass-fiber filters and are protected from lambda 5'-exonuclease, properties compatible with the presence of a topoisomerase II subunit bound to their 5' ends. Restriction enzyme cleavage of the fragments and hybridization with cloned alpha-globin-region probes reveal additional distinctive bands not seen in control DNA, allowing the localization of fragment ends near this gene cluster. The terminal regions of fragments from sucrose gradients or from field-inversion electrophoresis gels were also used to probe cloned regions of the gene cluster. Both approaches show that this cluster of three genes, which is not expressed in these cells, is located at a specific position in a approximately 20-kilobase DNA fragment. The upstream end of this fragment lies in a region that contains a site of DNA attachment to the nuclear matrix mapped by both in vivo and in vitro methods, and its downstream end is flanked by approximately 80% A + T sequences characteristic of matrix-attachment regions. These observations suggest that the DNA fragments are formed because topoisomerase II molecules can specifically and readily integrate into DNA at matrix-attachment regions and that the fragments represent entire DNA loops or domains.

Animals↗

PubMed2000

AHM1, a novel type of nuclear matrix-localized, MAR binding protein with a single AT hook and a J domain-homologous region.

Interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) have been implicated in various nuclear functions. We have identified a novel protein from wheat, AT hook-containing MAR binding protein1 (AHM1), that binds preferentially to MARs. A multidomain protein, AHM1 has the special combination of a J domain-homologous region and a Zn finger-like motif (a J-Z array) and an AT hook. For MAR binding, the AT hook at the C terminus was essential, and an internal portion containing the Zn finger-like motif was additionally required in vivo. AHM1 was found in the nuclear matrix fraction and was localized in the nucleoplasm. AHM1 fused to green fluorescent protein had a speckled distribution pattern inside the nucleus. AHM1 is most likely a nuclear matrix component that functions between intranuclear framework and MARs. J-Z arrays can be found in a group of (hypothetical) proteins in plants, which may share some functions, presumably to recruit specific Hsp70 partners as co-chaperones.

Amino Acid Sequence↗

PubMed1999

Interactions of Epstein-Barr virus origins of replication with nuclear matrix in the latent and in the lytic phases of viral infection.

Eukaryotic DNA is organized into domains or loops generated by the attachment of chromatin fibers to the nuclear matrix via specific regions called scaffold or matrix attachment regions. The role of these regions in DNA replication is currently under investigation since they have been found in close association with origins of replication. Also, viral DNA sequences, containing the origins of replication, have been found attached to the nuclear matrix. To investigate the functional role of this binding we have studied, in Raji cells, the interaction between Epstein-Barr virus (EBV) origins of replication and the nuclear matrix in relation to the viral cycle of infection. We report here that both the latent (ori P) and the lytic (ori Lyt) EBV origins of replication are attached to the nuclear matrix, the first during the latent cycle of infection and the second after induction of the lytic cycle. These findings suggest that the binding of the origins of replication with the nuclear matrix modulates viral replication and expression in the two different phases of infection.

Chromosome Mapping↗

PubMed2001

Sperm nuclear matrix association of the PRM1-->PRM2-->TNP2 domain is independent of Alu methylation.

Genes or multigenic chromosomal regions are organized by the nuclear matrix into a series of functionally discrete genic domains. Biophysical analysis of the human chromosome 16p13.13 region has shown that the PRM1-->PRM2-->TNP2 protamine containing multigenic locus is bounded by two sperm nuclear matrix attachment regions (MAR). This domain exists in a transcriptionally readied or potentiated (i.e. open) chromatin state when associated with the nuclear matrix. The MAR-bounded PRM1-->PRM2-->TNP2 locus is nestled in an Alu repetitive element dense region. Fluorescence in-situ hybridization, analysis of sperm nuclear matrix/halo preparations showed that the PRM1-->PRM2-->TNP2 domain specifically localizes to the sperm nuclear matrix. This raised the question of whether nuclear matrix association and gene expression in this locus is mediated by Alu methylation. The methylation status of the various Alu elements contained within the human PRM1-->PRM2-->TNP2 locus was therefore assayed. The seven Alu elements tested, including those associated with the matrix attachment regions within the PRM1-->PRM2-->TNP2 locus, were fully methylated in sperm DNA. Conversely, these same Alu repeats were hypomethylated within the erythroleukaemic cell line, K562, which does not express any of the genes from this domain. This study shows that Alu methylation status is independent of attachment of PRM1-->PRM2-->TNP2 locus to the nuclear matrix and that Alu methylation does not play a leading role in the regulation of this domain.

Alu Elements↗

PubMed2005

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

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

Antineoplastic Agents↗

PubMed2001

Hydrogen peroxide induces rapid digestion of oligodendrocyte chromatin into high molecular weight fragments.

High molecular weight (HMW) fragmentation of nuclear chromatin was studied in cultured rat oligodendrocytes (OL) exposed to hydrogen peroxide (H2O2). Intact genomic DNA was isolated by agarose embedding, and analyzed by field inversion gel electrophoresis, with and without S1 endonuclease digestion to detect and discriminate between single and double stranded fragmentations, respectively. The exposure of OL to H2O2 resulted in a very rapid degradation of chromosomal DNA into HMW fragments that reflect native chromatin structure. Hence, within 10 min after the addition of 1 mM H2O2, a discrete pool representing approximately 45% of the nuclear chromatin underwent single strand digestion into >400 kb fragments likely at AT-rich matrix attachment regions. Subsequent accumulation of single stand breaks at these regions led to bifilar scission. Ultimately, chromatin within this susceptible pool was cleaved at remaining matrix attachment regions into 50-200 kb fragments. Chromatin digestion could be elicited with H2O2 concentrations as low as 50 microM. After the removal of H2O2, most >400 kb fragments were religated within 2 h; however, digestion into 50-200 kb fragments was irreversible. The DNA digestion was not accompanied by the degradation of nuclear proteins, i.e., lamins A/C and poly (ADP-ribose) polymerase indicating that chromatin fragmentation is unlikely to be mediated by proteolysis. In conclusion, H2O2 at pathologically relevant concentrations induces a very rapid and extensive digestion of OL chromatin into HMW fragments. Because the chromatin fragmentation is only partly reversible, it may be a decisive factor in committing oxidatively stressed OL to degeneration and/or death.

Animals↗

PubMed2002

Tissue-specific association of the human tyrosine hydroxylase gene with the nuclear matrix.

Association of the human tyrosine hydroxylase (TH) gene with the nuclear matrix was studied using bovine matrices from tissues differing in expression of the gene. An in vitro binding assay was performed with fragments encompassing the -2300/+2300 region of the human TH gene. All fragments bound to the nuclear matrix from the adrenal medulla but none bound to that from the liver. A computer search of the sequenced part of the studied region revealed putative matrix attachment regions (S/MAR) - one in the promoter and two in the first intron. The (TCAT)(n) microsatellite repeat included in one of the intronic S/MARs seems to influence the binding potential of the human TH gene to the nuclear matrices.

Animals↗

PubMed1996

Analysis of hypermutation in immunoglobulin heavy chain passenger transgenes.

Somatic hypermutation of immunoglobulin (Ig) genes plays a critical role in the maturation of the human antibody response. The molecular basis of this important process is, however, unknown. To identify cis-acting sequences that initiate and target hypermutation, we have made three minitransgenes containing different portions of an Ig heavy chain (IgH) locus. Each transgene is a passenger, bearing a nonsense mutation preventing its translation; thus, transgene mutations reflect the endogenous mutational process and are not subject to affinity selection. To study transgenes after their circulation through the compartment associated with hypermutation in vivo, we rescued B cells as hybridomas after hyperimmunizing mice with the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). Hybridoma transgene and endogenous variable regions were amplified by polymerase chain reaction, subcloned, and sequenced. Endogenous anti-NP VDJ regions show the expected, at times extensive degree of base substitution. In mice bearing the smallest construct, which includes 2.4 kb of 5' IgH sequences, a rearranged VDJ region, the 5' matrix attachment region, and the intron enhancer, one of four evaluable hybridomas demonstrates two base substitutions in the V segment of one transgene copy. The two larger constructs include additional 3' IgH sequences (an alpha constant region and the 3' enhancer) and either the original VDJ segment or a substituted T cell receptor beta segment. Ten hybridomas derived from mice bearing these larger constructs demonstrate no evidence of targeted mutation, despite demonstrable transgene transcription in all hybridomas. In our system, mutation of a rearranged VDJ segment and surrounding promoter/enhancer regions is not increased by the juxtaposition of a constant region segment and the IgH 3' enhancer.

Animals↗

PubMed1995

Chromatin domains and prediction of MAR sequences.

Polynuceosomes are constrained into loops or domains and are insulated from the effects of chromatin structure and torsional strain from flanking domains by the cross-complexation of matrix-attached regions (MARs) and matrix proteins. MARs or SARs have an average size of 500 bp, are spaced about every 30 kb, and are control elements maintaining independent realms of gene activity. A fraction of MARs may cohabit with core origin replication (ORIs) and another fraction might cohabit with transcriptional enhancers. DNA replication, transcription, repair, splicing, and recombination seem to take place on the nuclear matrix. Classical AT-rich MARs have been proposed to anchor the core enhancers and core origins complexed with low abundancy transcription factors to the nuclear matrix via the cooperative binding to MARs of abundant classical matrix proteins (topoisomerase II, histone H1, lamins, SP120, ARBP, SATB1); this creates a unique nuclear microenvironment rich in regulatory proteins able to sustain transcription, replication, repair, and recombination. Theoretical searches and experimental data strongly support a model of activation of MARs and ORIs by transcription factors. A set of 21 characteristics are deduced or proposed for MAR/ORI sequences including their enrichment in inverted repeats, AT tracts, DNA unwinding elements, replication initiator protein sites, homooligonucleotide repeats (i.e., AAA, TTT, CCC), curved DNA, DNase I-hypersensitive sites, nucleosome-free stretches, polypurine stretches, and motifs with a potential for left-handed and triplex structures. We are establishing Banks of ORI and MAR sequences and have undertaken a large project of sequencing a large number of MARs in an effort to determine classes of DNA sequences in these regulatory elements and to understand their role at the origins of replication and transcriptional enhancers.

Animals↗

PubMed2004

Promoters and control elements: designing expression cassettes for gene therapy.

It has become apparent that the clinical success anticipated in the field of gene therapy has been limited by progress in several of the fundamental areas of genetics, molecular and cellular biology relevant to its application. Whilst a great deal of effort has been made in the evaluation of transgenes, it is only more recently with the advance of vector systems that attention has begun to be focused upon the means and control of transgene expression. Until recently, the majority of constructs have employed ubiquitous viral promoters to drive expression from simple gene expression cassettes using viral promoters and lacking introns, 3' untranslated regions (UTRs), locus control regions (LCR's), matrix attachment regions (MAR's) and other such genetic components. It has consequently emerged that these elements may have a key role in determining the levels and longevity of gene expression attainable in vivo, irrespective of the vector system utilised. The majority of gene therapy applications would also benefit from the specific optimisation of 'tailor-made' expression cassettes to optimise their therapeutic efficacy. In conjunction with modification of vector tropism and strategies to limit their immunogenicity, this should create vectors suitable for the clinical application of gene therapy. This review aims to highlight some of the principle considerations of gene expression in vivo, and the means by which it may most effectively be achieved, whether this is via the minimal modification of an existing eukaryotic promoter or by the more extensive design of a novel promoter and associated elements.

Animals↗

PubMed1997

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.

Amino Acid Sequence↗

PubMed1995

The role of immunoglobulin kappa elements in c-myc activation.

Burkitt's lymphoma cells are characterized by chromosomal translocations involving the proto-oncogene c-myc on chromosome 8 and one of the immunoglobulin gene loci on chromosome 2, 14 or 22. The translocated c-myc allele is transcriptionally activated, shows a preferential usage of promoter P1 over P2 (promoter shift) and lacks the ability to retain the transcription complex at the P2 promoter. In order to define the elements of the immunoglobulin kappa gene involved in deregulation of c-myc in a t(2;8) translocation, we designed constructs consisting of c-myc and different parts of the immunoglobulin kappa gene locus. Chromatin analysis of these stably transfected constructs revealed DNase I hypersensitive sites within the c-myc 5' region characteristic for an actively transcribed c-myc gene and three sites within the immunoglobulin kappa locus corresponding to the matrix attachment region, the intron and 3' enhancers, respectively. These three regulatory elements were necessary and sufficient for maximal c-myc activation and formation of the promoter shift. Kinetic nuclear run on experiments were performed to study the distribution of transcription complexes on c-myc exon 1 on constructs with and without the immunoglobulin kappa regulatory elements. The absence of a pausing polymerase complex at the c-myc P2 promoter could be demonstrated for constructs consisting of c-myc and the two kappa enhancers. Therefore the two enhancers are sufficient to relief the elongational block at the P2 promoter, however, the matrix attachment region is additionally required for maximal c-myc activation observed in Burkitt's lymphoma cells.

Base Sequence↗

PubMed1993

H-DNA can act as a transcriptional insulator.

The polypurine/polypyrimidine DNA stretches with repetitive sequences (H palindromes) can adopt triplex-mediated folded structures called H-DNA. H palindromes are more represented than expected for a random distribution of bases in eucaryotic genomes, suggesting that they could ensure some biological function. Most studies have focused attention on their possible involvement in the control of transcription because of their particularly high frequency in the 5'-flanking sequences of genes. Using the (5'-TTCCC-3')n sequence present in the upstream region of several genes, this work concludes to a novel potentiality of H palindromes: The strong ability to disrupt the cooperation between proximal elements initiating transcription and distal elements enhancing transcription over a long distance. We present three structural features of the TTCCC repeat likely to explain such an activity: (1) the visualization by electron microscopy showing that a long H palindrome spontaneously forms higher order tertiary structures; (2) the fact that this structure is a target for specific nuclear proteins displaying an affinity for single-stranded polypyrimidines; and (3) the preferential localization of the genomic TTCCC repetitive sequences at the level of chromosomal matrix attachment regions (MARs). We propose that H-DNA can insulate some genetic loci from influences of their chromosomal environment, and belongs to a subclass of genomic matrix attachment regions.

Animals↗

PubMed1995

Long-range fragmentation of the eukaryotic genome by exogenous and endogenous nucleases proceeds in a specific fashion via preferential DNA cleavage at matrix attachment sites.

Small cell lung cancer cells (OC-NYH-VM) were permeabilized and treated with different nucleases. The long-range distribution of DNA cleavage sites in the amplified c-myc gene locus was then analyzed by pulsed field gel electrophoretic separation of the released 50-kilobase to 1-megabase DNA fragments followed by indirect end labeling. Exogenous DNase I and nucleases specific for the single-stranded DNA were found to generate similar nonrandom patterns of large DNA fragments. The cleavage sites were located close to or even colocalized with matrix attachment regions, which were mapped independently using a recently developed procedure for DNA loop excision by DNA topoisomerase II-mediated DNA cleavage. Endogenous acidic nuclease with the properties of DNase II also digested DNA preferentially in proximity to the matrix attachment regions, generating characteristic patterns of excised DNA loops and their oligomers. A similar, although less specific, pattern of DNA fragmentation was observed after incubation of permeabilized cells under conditions favoring the activity of endogenous neutral Ca(2+)- and Mg(2+)-dependent nucleases. These findings are discussed in the context of the current model of the spatial domain organization of eukaryotic genome.

Calcium↗

PubMed2000

An upstream Oct-1- and Oct-2-binding silencer governs B29 (Ig beta) gene expression.

The B cell-specific B29 (Igbeta) gene is activated in the earliest B cell precursors and is expressed throughout B cell development. Tissue-specific expression of the murine B29 gene is controlled by a B cell-specific promoter whose activity is governed by a cassette of upstream transcriptional silencers. This study describes a potent new silencer that is located 5' of the previously identified B29 silencer elements, FROG and TOAD. Like these known elements, the new B29 silencer is not restricted to the B29 promoter. Nuclear proteins from all cell lines tested interacted with this A+T-rich sequence, which closely resembled a noncanonical octamer binding motif and also conformed to the consensus sequence for nuclear matrix attachment regions. Interaction of Oct-1 and Oct-2 with the B29 A+T-rich sequence was confirmed using octamer-specific Abs. Oct-1/Oct-2 binding was required for the inhibitory activity of this sequence because mutations that blocked Oct-1/Oct-2 binding also eliminated inhibition of the B29 promoter. This B29 A+T-rich sequence specifically interacted with isolated nuclear matrix proteins in vitro, suggesting that it may also function as a matrix attachment region element. Maintenance of the level of B29 gene expression through the interaction of the minimal promoter and the upstream silencer elements FROG, TOAD, and the A+T-rich Oct-1/Oct-2 binding motif may be essential for normal B cell development and/or function.

5' Untranslated Regions↗

PubMed2003

SUMO modification of a novel MAR-binding protein, SATB2, modulates immunoglobulin mu gene expression.

Nuclear matrix attachment regions (MARs) are regulatory DNA sequences that are important for higher-order chromatin organization, long-range enhancer function, and extension of chromatin modifications. Here we characterize a novel cell type-specific MAR-binding protein, SATB2, which binds to the MARs of the endogenous immunoglobulin micro locus in pre-B cells and enhances gene expression. We found that SATB2 differs from the closely related thymocyte-specific protein SATB1 by modifications of two lysines with the small ubiquitive related modifier (SUMO), which are augmented specifically by the SUMO E3 ligase PIAS1. Mutations of the SUMO conjugation sites of SATB2 enhance its activation potential and association with endogenous MARs in vivo, whereas N-terminal fusions with SUMO1 or SUMO3 decrease SATB2-mediated gene activation. Sumoylation is also involved in targeting SATB2 to the nuclear periphery, raising the possibility that this reversible modification of a MAR-binding protein may contribute to the modulation of subnuclear DNA localization.

B-Lymphocytes↗

PubMed2006

Functional interaction between PML and SATB1 regulates chromatin-loop architecture and transcription of the MHC class I locus.

The function of the subnuclear structure the promyelocytic leukaemia (PML) body is unclear largely because of the functional heterogeneity of its constituents. Here, we provide the evidence for a direct link between PML, higher-order chromatin organization and gene regulation. We show that PML physically and functionally interacts with the matrix attachment region (MAR)-binding protein, special AT-rich sequence binding protein 1 (SATB1) to organize the major histocompatibility complex (MHC) class I locus into distinct higher-order chromatin-loop structures. Interferon gamma (IFNgamma) treatment and silencing of either SATB1 or PML dynamically alter chromatin architecture, thus affecting the expression profile of a subset of MHC class I genes. Our studies identify PML and SATB1 as a regulatory complex that governs transcription by orchestrating dynamic chromatin-loop architecture.

Cell Line↗

PubMed2000

Caught in the act: binding of Ku and PARP to MARs reveals novel aspects of their functional interaction.

Specific regions of eukaryotic genomic DNA that exhibit high-affinity binding to the nuclear matrix in vitro are called matrix attachment regions (MARs) and are implicated in the loop domain organization of chromatin. Small regions possessing high base unpairing potential within these MARs are referred to as base unpairing regions (BURs). BUR-affinity chromatographic separations of proteins from breast cancer cells yielded, almost exclusively, a mixture of poly (ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK), two nuclear enzymes that are implicated in the cellular response to DNA damage. Contrary to the long-held notion that PARP and Ku autoantigen, the DNA-binding heterodimeric subunit of DNA-PK, bind only to DNA ends, recently we have shown that both proteins individually bind BURs with high affinity and specificity in an end-independent manner. Furthermore, Ku autoantigen forms a molecular complex with PARP in the absence of DNA, and the physical association of these proteins synergistically enhanced their BUR-binding activity. Autoribosylation of PARP abolished its association with Ku autoantigen and BUR-binding activity. These findings have, for the first time, provided a molecular link toward elucidating the functional interaction between PARP and DNA-PK. The identification of MARs as their common binding target suggests a novel role for these enzymes in the modulation of chromatin structure and function.

Antigens, Nuclear↗