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Results for “Matrix Attachment Regions”

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PubMed1996

Histone modifications, chromatin structure, and the nuclear matrix.

The nuclear matrix has a role in the organization and function of nuclear DNA. A combination of stable and transient interactions between chromatin and the nuclear matrix is involved in organizing DNA within the nucleus. DNA sequences (matrix attachment regions) at the base of a loop bind to nuclear matrix proteins and arrange the nuclear DNA into chromatin loop domains. Multiple, transient interactions between the nuclear matrix and transcriptionally active chromatin are thought to be responsible for the insoluble feature of transcriptionally active chromatin. Current evidence suggests that histone acetyltransferase, histone deacetylase (enzymes that catalyze rapid histone acetylation and deacetylation), transcription factors, and the transcription machinery mediate the transient attachments between nuclear matrix and active chromatin. Highly acetylated core histones, which are associated with transcriptionally active DNA, are also ubiquitinated and phosphorylated. Recent studies show that specific H1 subtypes and their phosphorylated isoforms are localized in centers of RNA splicing in the nucleus. The implications of these findings and the impact of the histone modifications on the nuclear-organization of chromatin are discussed.

Animals↗

PubMed1995

Recurrence of WHV integration in the b3n locus in woodchuck hepatocellular carcinoma.

Frequent occurrence of woodchuck hepatitis virus DNA (WHV DNA) integration into or in proximity to myc oncogenes and in the win locus of cellular genome in woodchuck hepatocellular carcinomas (HCC) has been described by several authors. We report a further cellular locus as a recurrent target for WHV integration in woodchuck HCCs. A WHV DNA integration and its cellular flanking regions were cloned from a HCC developed in a chronically WHV-infected woodchuck. Sequence analysis showed integration of rearranged C, PreS1, and 5' truncated X regions of the WHV genome, located in a cellular locus previously described for WHV integration in another woodchuck HCC. The two integration sites are only about 0.5 kb apart. In addition to Alu-like repeats and a gag-like coding region, previously described, we found several features of MAR (matrix attachment region) chromosomal sequences in the normal cellular locus, leading us to predict that part of it might be a previously unrecognized MAR.

Animals↗

PubMed2001

AT-rich islands in genomic DNA as a novel target for AT-specific DNA-reactive antitumor drugs.

Interstrand cross-links at T(A/T)4A sites in cellular DNA are associated with hypercytotoxicity of an anticancer drug, bizelesin. Here we evaluated whether these lethal effects reflect targeting critical genomic regions. An in silico analysis of human sequences showed that T(A/T)4A motifs are on average scarce and scattered. However, significantly higher local motif densities were identified in distinct minisatellite regions (200-1000 base pairs of approximately 85-100% AT), herein referred to as "AT islands." Experimentally detected bizelesin lesions agree with these in silico predictions. Actual bizelesin adducts clustered within the model AT island naked DNA, whereas motif-poor sequences were only sparsely adducted. In cancer cells, bizelesin produced high levels of lesions (approximately 4.7-7.1 lesions/kilobase pair/microM drug) in several prominent AT islands, compared with markedly lower lesion levels in several motif-poor loci and in bulk cellular DNA (approximately 0.8-1.3 and approximately 0.9 lesions/kilobase pair/microM drug, respectively). The identified AT islands exhibit sequence attributes of matrix attachment regions (MARs), domains that organize DNA loops on the nuclear matrix. The computed "MAR potential" and propensity for supercoiling-induced duplex destabilization (both predictive of strong MARs) correlate with the total number of bizelesin binding sites. Hence, MAR-like AT-rich non-coding domains can be regarded as a novel class of critical targets for anticancer drugs.

Adenine↗

PubMed2002

The chicken lysozyme chromatin domain contains a second, widely expressed gene.

The chicken lysozyme (cLys) locus has been shown to contain all of the cis-elements necessary for position-independent and tissue-specific expression entirely within a 24-kb region defined by general DNase I sensitivity and flanked by matrix attachment regions. As such, it has been viewed as an example of a functional chromatin domain, which is structurally and functionally isolated from neighbouring chromatin. We report here the identification and characterisation of the chicken glioma-amplified sequence (cGas41) locus, which though widely expressed, is contained entirely within the lysozyme chromatin domain. The cGas41 transcript encodes a putative transcription factor, starts 207 bp downstream of the cLys polyadenylation site and is preceded by a CpG island with proposed dual promoter/origin function. The location and differential expression of cGas41 compels re-evaluation of the accumulated literature on the lysozyme domain, and represents an example of two unrelated, differentially expressed vertebrate genes coexisting in the same functional chromatin domain.

Amino Acid Sequence↗

PubMed1998

Attachment to the nuclear matrix mediates specific alterations in chromatin structure.

The DNA in eukaryotic chromosomes is organized into a series of loops that are permanently attached at their bases to the nuclear scaffold or matrix at sequences known as scaffold-attachment or matrix-attachment regions. At present, it is not clear what effect affixation to the nuclear matrix has on chromatin architecture in important regulatory regions such as origins of replication or the promoter regions of genes. In the present study, we have investigated cell-cycle-dependent changes in the chromatin structure of a well characterized replication initiation zone in the amplified dihydrofolate reductase domain of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Replication can initiate at any of multiple potential sites scattered throughout the 55-kilobase intergenic region in this domain, with two subregions (termed ori-beta and ori-gamma) being somewhat preferred. We show here that the chromatin in the ori-beta and ori-gamma regions undergoes dramatic alterations in micrococcal nuclease hypersensitivity as cells cross the G1/S boundary, but only in those copies of the amplicon that are affixed to the nuclear matrix. In contrast, the fine structure of chromatin in the promoter of the dihydrofolate reductase gene does not change detectably as a function of matrix attachment or cell-cycle position. We suggest that attachment of DNA to the nuclear matrix plays an important role in modulating chromatin architecture, and this could facilitate the activity of origins of replication.

Animals↗

PubMed2004

Comparative analysis of the PDCD2-TBP-PSMB1 region in vertebrates.

Three orthologous genes encoding programmed cell death 2 (PDCD2), TATA-binding protein (TBP), and proteasomal subunit C5 (PSMB1) proteins have been shown previously to be nonrandomly distributed in both mammalian and invertebrate genomes. Here we analyze a conserved synteny of the PDCD2, TBP, and PSMB1 orthologs in four nonmammalian vertebrates. Homologous genes of the chicken, zebrafish, fugu, and Tetraodon nigroviridis were identified. A chicken cosmid harboring the orthologs of these three genes was completely sequenced. The fish genes were analyzed in silico. In all seven vertebrates thus far investigated, the PDCD2 and TBP genes are located tail-to-tail. In six tested species but the zebrafish, the PSMB1 gene mapped head-to-head or in the close vicinity to the TBP, but even in the zebrafish, all three genes were syntenic. In contrast, a three times reused synteny breakpoint in the 5'-region from PDCD2 was detected. A comparative analysis revealed the distribution of putative matrix-attached regions (MARs), which may affect the synteny conservation.

Animals↗

PubMed1998

The effects of E mu, 3'alpha (hs 1,2) and 3'kappa enhancers on mutation of an Ig-VDJ-Cgamma2a Ig heavy gene in cultured B cells.

The Ig kappa intronic and 3'kappa light chain enhancers have been shown to be necessary for V region hypermutation in kappa light chain transgenes. To investigate the role of the E mu intronic enhancer in V region hypermutation of heavy chain genes, E mu and its associated matrix attachment regions (MAR) were replaced with the SV40 or the cytomegalovirus (CMV) enhancer in a gamma2a construct that hypermutates its rearranged VDJ region in the NSO plasmacytoma and 18.81 pre-B cell lines. In this model in vitro system, mutation rates of stable transfectants were determined by reversion analysis using a V region stop codon that, when mutated, allowed the detection of cellular revertants by ELISA spot assay. The gamma2a constructs with the E mu, SV40 and CMV enhancers mutated at comparably high rates in the B cell lines, but not in L cells, indicating that the E mu enhancer and its associated MAR were not specifically required for IgH hypermutation in this system. In parallel experiments, the addition of the 3'alpha heavy chain enhancer (hs 1,2) or the 3'kappa light chain enhancer did not increase the mutation rate of a related mu reporter construct in which the associated VDJ mutates at a moderately low rate in NSO cells or in cell hybrids made between 18.81 and NSO. These results imply that cis-acting IgH elements that promote hypermutation may not be restricted to Ig-specific transcriptional enhancers.

Animals↗

PubMed2000

Differential regulation of gene activity and chromatin structure within the human serpin gene cluster at 14q32.1 in macrophage microcell hybrids.

The human gene encoding alpha1-antitrypsin (alpha1AT, gene symbol PI ) is highly expressed in the liver and in cultured hepatoma cells and, to a lesser extent, in macrophages, where transcription originates from a separate upstream promoter. alpha1AT maps to a region of human chromosome 14q32.1 that includes a related serine protease inhibitor (serpin) gene that encodes corticosteroid-binding globulin (CBG). We recently reported the chromatin organization of this approximately 130 kb region, as defined by DNase I hypersensitive sites (DHSs) and matrix-attachment regions, in expressing and non-expressing cells. Furthermore, we demonstrated that transfer of human chromosome 14 from non-expressing fibroblasts to rat hepatoma cells resulted in activation of both alpha1AT and CBG transcription and gene activation was accompanied by long range chromatin reorganization of the entire region. In this study, we transferred human chromosome 14 from fibroblasts to mouse macrophages and documented activation of alpha1AT but not CBG gene expression. RT-PCR experiments indicated that transcription of the human alpha1AT gene in the microcell hybrids initiated at the macrophage promoter. Furthermore, DHS mapping experiments revealed a distinctive chromatin configuration of the locus that resembled the structure found in human macrophage-like cell lines, with many DHSs around alpha1AT but few in CBG. Thus, mouse macrophage cell lines will provide a useful cell type to study the effects of targeted modifications of the human alpha1AT-CBG locus on the regulation of cell-specific gene activity and chromatin structure.

Animals↗

PubMed2006

The H19 gene: regulation and function of a non-coding RNA.

The H19 gene encodes a 2.3-kb non-coding mRNA which is strongly expressed during embryogenesis. This gene belongs to an imprinted cluster, conserved on mouse chromosome 7 and human chromosome 11p15. H19 is maternally expressed and the neighbouring Igf2 gene is transcribed from the paternal allele. These two genes are co-expressed in endoderm- and mesoderm-derived tissues during embryonic development, which suggests a common mechanism of regulation. The regulatory elements (imprinted control region, CTCF insulation, different enhancer sequences, promoters of the two genes, matrix attachment regions) confer a differential chromatin architecture to the two parental alleles leading to reciprocal expression. The role of the H19 gene is unclear but different aspects have been proposed. H19 influences growth by way of a cis control on Igf2 expression. Although H19(-/-) mice are viable, a role for this gene during development has been suggested by viable H19(-/-) parthenogenetic mice. Finally it has been described as a putative tumour suppressor gene. H19 has been studied by numerous laboratories over the last fifteen years, nevertheless the function of this non-coding RNA remains to be elucidated.

Animals↗

PubMed2003

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

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

Animals↗

PubMed2002

Initiation sites are distributed at frequent intervals in the Chinese hamster dihydrofolate reductase origin of replication but are used with very different efficiencies.

Previous radiolabeling and two-dimensional (2-D) gel studies of the dihydrofolate reductase (DHFR) domain of Chinese hamster cells have suggested that replication can initiate at any one of a very large number of inefficient sites scattered throughout the 55-kb intergenic spacer region, with two broad subregions (ori-beta and ori-gamma) preferred. However, high-resolution analysis by a PCR-based nascent strand abundance assay of the 12-kb subregion encompassing ori-beta has suggested the presence of a relatively small number of fixed, highly efficient initiation sites distributed at infrequent intervals that correspond to genetic replicators. To attempt to reconcile these observations, two different approaches were taken in the present study. In the first, neutral-neutral 2-D gel analysis was used to examine replication intermediates in 31 adjacent and overlapping restriction fragments in the spacer, ranging in size from 1.0 to 18 kb. Thirty of 31 fragments displayed the complete bubble arcs characteristic of centered origins. Taking into account overlapping fragments, these data suggest a minimum of 14 individual start sites in the spacer. In the second approach, a quantitative early labeled fragment hybridization assay was performed in which radioactive origin-containing DNA 300 to 1,000 nucleotides in length was synthesized in the first few minutes of the S period and used to probe 15 clones distributed throughout the intergenic spacer but separated on average by more than 1,000 bp. This small nascent DNA fraction hybridized to 14 of the 15 clones, ranging from just above background to a maximum at the ori-beta locus. The only silent region detected was a small fragment lying just upstream from a centered matrix attachment region--the same region that was also negative for initiation by 2-D gel analysis. Results of both approaches suggest a minimum of approximately 20 initiation sites in the spacer (two of them being ori-beta and ori-gamma), with ori-beta accounting for a maximum of approximately 20% of initiations occurring in the spacer. We believe that the results of all experimental approaches applied to this locus so far can be fitted to a model in which the DHFR origin consists of a 55-kb intergenic zone of potential sites that are used with very different efficiencies and which are separated in many cases by a few kilobases or less.

Animals↗

PubMed1994

A compilation and classification of DNA binding sites for protein transcription factors from vertebrates.

The field of protein transcription regulators and their DNA sequence specificity has been the most rapidly expanding in the last few years. The concerted interplay of protein transcription factors on the regulatory regions of eukaryotic genes (promoters, enhancers, origins of replication, silencers, and matrix-attached regions) regulates transcription levels; the differential activity of genes during development and the cell cycle, between cell types, and in response to physiological stimuli results from interdigitation of regulatory circuits controlling transcription initiation, finely tuned by the relative amounts of protein factors synthesized in a cell type, their phosphorylation, isoforms within factor families, the way protein regulators are brought in contact with one another through the patchwork of their cognate sites on the regulatory regions of genes, and by regulation of their nuclear import. The varying affinity of the same factor for its cognate DNA in different promoters can also be modulated by the type of proteins it is brought into contact with, by one or more nucleotide changes in its binding sites among promoters, and by the chromatin structure. The classification of protein transcriptional regulators attempted here according to their DNA binding specificity into those that bind AT-, GC-, GA, TG-rich and mixed motif has one obvious advantage: different protein factors that bind to the same DNA sequence will be found within the same class. In addition, this classification has allowed us to discern a class of transcriptional regulators whose binding site consists of a GA- and a CT-rich moiety; no other two pairs of dinucleotides compose a major class of factor sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2002

The interaction between the human beta-globin locus control region and nuclear matrix.

Our previous study showed that hydroxyurea (Hu) could induce HEL cells to express human beta-globin gene. However the molecular mechanisms by which the expression of beta-globin gene is activated and regulated are poorly understood. Here we show that the binding patterns between the core DNA sequences (HS2 core sequence -10681 approximately -10971 bp, HS3 core sequence -14991 approximately -14716 bp and HS4 core sequence -18586 approximately -18306 bp) of DNase I hypersensitive sites in the human beta-globin LCR and nuclear matrix proteins isolated from Hu induced and uninduced HEL cells are quite different. Results demonstrated that nuclear matrix proteins might play important roles in regulating the expression of human beta-like globin genes through their interaction with HSs (HS2, HS3 and HS4 core sequences) in the LCR. Moreover, the results obtained from the in vitro DNA-matrix binding assay showed that the core DNA sequences of DNase I hypersensitive sites (HS2, HS3 and HS4) were unable to bind to the nuclear matrix isolated from uninduced HEL cells; in addition, HS2 core DNA sequence was capable of binding to the nuclear matrix prepared from Hu-induced HEL cells, while both HS3 and HS4 core DNA sequences could not do so. Results indicated that the HS2 core DNA sequence may be a functional MAR (matrix attachment region). We suggest that the HS2 core DNA sequence binding to the nuclear matrix in Hu-induced HEL cells may open the structure of chromatin to make the LCR accessible to the promoter of beta-globin gene and to promote its transcription.

Binding Sites↗

PubMed2004

Replication initiation in mammalian cells: changing preferences.

Accurate duplication of genetic material is central to cell proliferation. In eukaryotes, S phase is tightly controlled during development. For example, initiation events occur at random sites in early embryos but later appear restricted to preferred DNA regions. Epigenetic changes depending on chromatin organization and/or availability of specific factors likely control origin choice. By using the dynamic molecular combing technology, coupled with specific labeling of neo-synthesized DNA and FISH, we recently demonstrated that the efficiency and spacing of initiation sites are still flexible in mammalian somatic cells, and strongly rely on nucleotide availability. In all conditions, initiation events appear confined to short AT-rich sequences previously identified as matrix attachment regions, which suggests a direct involvement of these features in origin specification. Functional relationships between matrix anchorage and origin selection are discussed.

Animals↗

PubMed1990

A nuclear matrix protein binds very tightly to DNA in the avian beta-globin gene enhancer.

Current evidence suggests that DNA is covalently attached to proteins in the nuclear matrix of eukaryotic cells and that specific DNA sequences are tightly associated with the nuclear matrix. However, it has not been documented that specific DNA sequences can become covalently attached to nuclear matrix protein. We have examined the binding of cloned DNA sequences that contain the avian beta-globin gene enhancer, a region previously shown to be matrix associated in erythroid cells in vivo, with nuclear matrices from several avian tissue sources to determine if covalent DNA-protein bonds are formed. Our results indicate that sequence-specific DNA-protein complexes that are resistant to denaturation by SDS, boiling, and phenol and disulfide reduction are formed. Excess protein, capable of forming very tight bonds with DNA that contains the beta-globin gene enhancer, is present in cells in which matrix attachment of this DNA sequence is not detected in vivo. Evidence is presented that suggests that the protein to which DNA forms very tight bonds is not topoisomerase II. These results are discussed in relation to current models of the nuclear matrix and the utility of in vitro assays of matrix attachment regions using cloned DNA.

Animals↗

PubMed1995

Nuclear matrix acceptor binding sites for steroid hormone receptors: a candidate nuclear matrix acceptor protein.

Steroid/nuclear-hormone receptors are ligand-activated transcription factors that have been localized to the nuclear matrix. The classic model of hormone action suggests that, following activation, these receptors bind to specific "steroid response elements" on the DNA, then interact with other factors in the transcription initiation complex. However, evidence demonstrates the existence of specific chromatin proteins that act as accessory factors by facilitating the binding of the steroid receptors to the DNA. One such protein, the "receptor binding factor (RBF)-1", has been purified and shown to confer specific, high-affinity binding of the progesterone receptor to the DNA. Interestingly, the RBF-1 is localized to the nuclear matrix. Further, the RBF-1 binds specifically to a sequence of the c-myc proto-oncogene that has the appearance of a nuclear matrix attached region (MAR). These results, and other findings reviewed here, suggest that the nuclear matrix is involved intimately in steroid hormone-regulated gene expression.

Animals↗

PubMed1996

A nuclear matrix-specific factor that binds a specific segment of the negative regulatory element (NRE) of HIV-1 LTR and inhibits NF-kappa(B) activity.

The negative regulatory element (NRE) of human immunodeficiency virus type-1 (HIV-1) long terminal repeat (LTR) is a defined region that has been reported to downregulate LTR-directed HIV gene expression. However, information on the precise role of this region in regulating HIV gone transcription is lacking. We have investigated the possibility that these NRE sequences regulate HIV transcription by a mechanism mediated through a nuclear matrix-specific DNA-protein interaction. We find a nuclear matrix attachment region (MAR) present within the NRE of the HIV-1 LTR that recognizes a sequence-specific DNA-binding protein present in the nuclear matrix of HIV infected cells. Moreover, we also show that the purified DNA-binding nuclear matrix protein (NMP) specifically represses the DNA-binding activity of NF-kappaB. It is likely that the MAR and MAR-enriched specific DNA-binding NMP are brought into juxtaposition by the non-chromatin scaffolding of the nucleus, thus influencing NF-kappaB (and other nuclear proteins) DNA-binding activity through protein-protein and protein-DNA interactions. Our date suggest that one possible role of the NRE could be to act as a matrix attachment site in the nuclear matrix, thus, allowing interaction with a sequence-specific trans-acting factor. The negative effect on NF-kappaB activity due to this MAR-NMP-specific interaction provides a mechanism by which the NRE downregulates HIV gene expression.

Antigens, Nuclear↗

PubMed2001

Target sites for SINE integration in Brassica genomes display nuclear matrix binding activity.

Short interspersed nuclear elements (SINEs) are ubiquitous components of complex animal and plant genomes. SINEs are believed to be important players in eukaryotic genome evolution. Studies on SINE integration sites have revealed non-random integration without strict nucleotide sequence requirements for the integration target, suggesting that the targeted DNA might assume specific secondary structures or protein associations. Here, we report that S1 SINE elements in the genomes of Brassica show an interesting preference for matrix attachment regions (MARs). Ten cloned genomic regions were tested for their ability to bind the nuclear matrix both before and after a SINE integration event. Eight of the genomic regions targeted by S1 display strong affinity for the nuclear matrix, while two show weaker binding. The SINE S1 did not display any matrix-binding capacity on its own in either non-methylated or methylated forms. In vivo, an integrated S1 is methylated while the surrounding genomic regions may remain undermethylated or undergo methylation. However, tested genomic regions containing methylated S1, with or without methylated flanking genomic sequences, were found to vary in their ability to bind the matrix in vitro. These results suggest a possible molecular basis for a preferential targeting of SINEs to MARs and a possible impact of the integration events upon gene and genome function.

Binding, Competitive↗