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Results for “Matrix Attachment Regions”

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PubMed2002

Synergistic induction of the senescence-associated genes by 5-bromodeoxyuridine and AT-binding ligands in HeLa cells.

5-Bromodeoxyuridine induces a senescence-like phenomenon in mammalian cells. This effect was dramatically potentiated by AT-binding ligands such as distamycin A, netropsin, and Hoechst 33258. The genes most remarkably affected by these ligands include the widely used senescence-associated genes and were located on or nearby Giemsa-dark bands of human chromosomes. We hypothesize that AT-rich scaffold/nuclear matrix attachment region sequences are involved in this phenomenon. In fact, upon substitution of thymine with 5-bromouracil, a rat S/MAR sequence reduced its degree of bending and became insensitive to cancellation of the bending by distamycin A. The S/MAR sequence containing 5-bromouracil also bound more tightly to nuclear scaffold proteins in vitro and this binding was not inhibited by distamycin A. Under the same conditions, the S/MAR sequence containing thymine easily dissociated from the nuclear scaffold proteins. Taken together, the synergistic induction of the genes may be explained not only by opening of condensed chromatin by distamycin A but also by increase in the binding of 5-bromouracil-containing S/MAR sequences to the nuclear scaffolds.

AT Rich Sequence↗

PubMed1996

A role for nuclear NF-kappaB in B-cell-specific demethylation of the Igkappa locus.

The immunoglobulin kappa gene is specifically demethylated during B-cell maturation in a process which utilizes discrete cis-acting modules such as the intronic kappa enhancer element and the matrix attachment region (MAR). While any MAR sequence is sufficient for this reaction, mutation analysis indicates that tissue specificity is mediated by kappaB binding sequences within the kappa intronic enhancer. The plasmacytoma cell line S107 lacks kappaB binding activity and fails to demethylate the kappa locus. However, B-cell specific demethylation is restored by the introduction of an active kappaB binding protein gene relB. This represents the first demonstration of a trans-acting factor involved in cell-type-specific demethylation, and suggests that the same protein-DNA recognition system used for transcription may also contribute to the earlier developmental events that bring about activation of the kappa locus.

B-Lymphocytes↗

PubMed1998

PTH-responsive osteoblast nuclear matrix architectural transcription factor binds to the rat type I collagen promoter.

In connective tissue, cell structure contributes to type I collagen expression. Differences in osteoblast microarchitecture may account for the two distinct cis elements regulating basal expression, in vivo and in vitro, of the rat type I collagen alpha1(I) polypeptide chain (COL1A1). The COL1A1 promoter conformation may be the penultimate culmination of osteoblast structure. Architectural transcription factors bind to the minor groove of AT-rich DNA and bend it, altering interactions between other trans-acting proteins. Similarly, nuclear matrix (NM) proteins bind to the minor groove of AT-rich matrix-attachment regions, regulating transcription by altering DNA structure. We propose that osteoblast NM architectural transcription factors link cell structure to promoter geometry and COL1A1 transcription. Our objective was to identify potential osteoblast NM architectural transcription factors near the in vitro and in vivo regulatory regions of the rat COL1A1 promoter. Nuclear protein-promoter interactions were analyzed by gel shift analysis and related techniques. NM extracts were derived from rat osteosarcoma cells and from rat bone. The NM protein, NMP4, and a soluble nuclear protein, NP, both bound to two homologous poly(dT) elements within the COL1A1 in vitro regulatory region and proximal to the in vivo regulatory element. These proteins bound within the minor groove and bent the DNA. Parathyroid hormone increased NP/NMP4 binding to both poly(dT) elements and decreased COL1A1 mRNA in the osteosarcoma cells. NP/NMP4-COL1A1 promoter interactions may represent a molecular pathway by which osteoblast structure is coupled to COL1A1 expression.

Animals↗

PubMed1993

Multitude of inverted repeats characterizes a class of anchorage sites of chromatin loops to the nuclear matrix.

In order to understand the nature of DNA sequences that organize chromatin into domains or loops, we have cloned the nuclear matrix DNA (1.7% of the total DNA) from human myelogenous leukemia cells in culture. Nuclear matrix is formed by interactions between specific stretches of DNA of about 0.1 to 5.0 kb with protein transcription factors, nuclear enzymes, and structural proteins. Nuclear matrix is believed to be the exclusive nuclear microenvironment in which initiation of DNA replication, transcription, and repair take place. The matrix attachment regions (MARs) of DNA have transcriptional enhancer activity, harbor the origins of replication of the human genome, and define the borders between neighboring chromatin loops. In this study we report the sequence of the human MAR fragment 19.2 of a size of 542 bp. Hum. MAR 19.2 is composed of TG-, CA-, CT-, and GA-rich blocks and shows 8 perfect and imperfect inverted repeats. Thus, we have identified a novel class of MARs with sequence characteristics divergent from the AT-rich class of MARs. The inverted repeats of the 19.2 sequence might be stabilized into their cruciform configuration by torsional strain and by specific transcription/replication protein factors. This MAR might function in the initiation of replication of the flanking chromatin domain and in the regulation of the transcriptional activity of the gene(s) that reside in this domain.

Base Sequence↗

PubMed2006

[Nuclear organization and expression of milk protein genes].

Milk protein gene expression varies during the pregnancy/lactation cycle under the influence of lactogenic hormones which induce the activation of several transcription factors. Beyond this activation modifying the binding properties of these factors to their consensus sequences, their interactions with DNA is regulated by variations of the chromatin structure. In the nuclei of the mammary epithelial cell, the three dimensional organisation of the chromatin loops, located between matrix attachment regions, is now being studied. The main milk components are organised in supramolecular structures. Milk fat globules are made of a triglyceride core enwrapped by a tripartite membrane originating from various intracellular compartments. The caseins, the main milk proteins, form aggregates: the casein micelles. Their gradual aggregation in the secretory pathway is initiated as soon as from the endoplasmic reticulum. The mesostructures of the milk fat globule and of the casein micelle remain to be elucidated. Our goal is to make some progress into the understanding of the molecular and cellular mechanisms involved in the formation of these milk products.

Animals↗

PubMed1996

c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1.

Two genetic events contribute to the development of endemic Burkitt lymphoma (BL) infection of B lymphocytes with Epstein-Barr virus (EBV) and the activation of the protooncogene c-myc through chromosomal translocation. The viral genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for transformation of primary human B cells by EBV in vitro; however, these genes are not expressed in BL cells in vivo. To address the question whether c-myc activation might abrogate the requirement of the EBNA2 and LMP1 function, we have introduced an activated c-myc gene into an EBV-transformed cell line in which EBNA2 was rendered estrogen-dependent through fusion with the hormone binding domain of the estrogen receptor. The c-myc gene was placed under the control of regulatory elements of the immunoglobulin kappa locus composed a matrix attachment region, the intron enhancer, and the 3' enhancer. We show here that transfection of a c-myc expression plasmid followed by selection for high MYC expression is capable of inducing continuous proliferation of these cells in the absence of functional EBNA2 and LMP1. c-myc-induced hormone-independent proliferation was associated with a dramatic change in the growth behavior as well as cell surface marker expression of these cells. The typical lymphoblastoid morphology and phenotype of EBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern of viral and cellular genes rather than its germinal center origin.

Antigens, CD↗

PubMed1999

[Identification and mapping on human chromosome 19 of chromosome-specific repeated element preferentially binding with nuclear matrix].

From a library of sequences binding preferentially to nuclear matrix (matrix attachment regions, MARs), a fragment of about 300 bp in length (CEA (carcinoembryonic antigen)-MAR) was isolated and characterized. The CEA-MAR sequence was found in more than ten loci of chromosome 19 containing elements similar to genes of the CEA family. No sequences of this group were found on other human chromosomes. Two CEA-MAR-containing loci were sequenced, and sequences for another seven loci were found in GenBank. A comparative analysis of CEA-MARs and the flanking sequences is reported. Based on the sequence of the CEA-containing chromosome 19 loci, a hypothetical model of the domain structure of a 2-Mb chromosome region was constructed and the mutual arrangement of CEA-MARs and genes of CEA family was elucidated. The CEA-MARs were located 5-20 kb downstream of the CEA genes. These results suggest that the duplication unit of the CEA family may coincide with chromatin domains containing these genes.

Base Sequence↗

PubMed2003

Interaction in vitro of type III intermediate filament proteins with Z-DNA and B-Z-DNA junctions.

The selection of DNA fragments containing simple d(GT)(n) and composite d(GT)(m). d(GA)(n) microsatellites during affinity binding of mouse genomic DNA to type III cytoplasmic intermediate filaments (cIFs) in vitro, and the detection of such repeats, often as parts of nuclear matrix attachment region (MAR)-like DNA, in SDS-stable DNA-vimentin crosslinkage products isolated from intact fibroblasts, prompted a detailed study of the interaction of type III cIF proteins with left-handed Z-DNA formed from d(GT)(17) and d(CG)(17) repeats under the topological tension of negatively supercoiled plasmids. Although d(GT)(n) tracts possess a distinctly lower Z-DNA-forming potential than d(CG)(n) tracts, the filament proteins produced a stronger electrophoretic mobility shift with a plasmid carrying a d(GT)(17) insert than with plasmids containing different d(CG)(n) inserts, consistent with the facts that the B-Z transition of d(GT)(n) repeats requires a higher negative superhelical density than that of d(CG)(n) repeats and the affinity of cIF proteins for plasmid DNA increases with its superhelical tension. That both types of dinucleotide repeat had indeed undergone B-Z transition was confirmed by S1 nuclease and chemical footprinting analysis of the plasmids, which also demonstrated efficient protection by cIF proteins from nucleolytic and chemical attack of the Z-DNA helices as such, as well as of the flanking B-Z junctions. The analysis also revealed sensibilization of nucleotides in the center of one of the two strands of a perfect d(CG)(17) insert toward S1 nuclease, indicating cIF protein-induced bending of the repeat. In all these assays, vimentin and glial fibrillary acidic protein (GFAP) showed comparable activities, versus desmin, which was almost inactive. In addition, vimentin and GFAP exhibited much higher affinities for the Z-DNA conformation of brominated, linear d(CG)(25) repeats than for the B-DNA configuration of the unmodified oligonucleotides. While double-stranded DNA was incapable of chasing the Z-DNA from its protein complexes, and Holliday junction and single-stranded (ss)DNA were distinguished by reasonable competitiveness, phosphatidylinositol (PI) and, particularly, phosphatidylinositol 4,5-diphosphate (PIP(2)) turned out to be extremely potent competitors. Because PIP(2) is an important member of the nuclear PI signal transduction cascade, it might exert a regulatory influence on the binding of cIF proteins to Z- and other DNA conformations. From this interaction of cIF proteins with Z- and bent DNA and their previously detected affinities for MAR-like, ss, triple helical, and four-way junction DNA, it may be concluded that the filament proteins play a general role in such nuclear matrix-associated processes as DNA replication, recombination, repair, and transcription.

Animals↗

PubMed1996

Common structural features of replication origins in all life forms.

Origins of replication (ORIs) among prokaryotes, viruses, and multicellular organisms appear to possess simple tri-, tetra-, or higher dispersed repetitions of nucleotides, AT tracts, inverted repeats, one to four binding sites of an initiator protein, intrinsically curved DNA, DNase I-hypersensitive sites, a distinct pattern of DNA methylation, and binding sites for transcription factors. Eukaryotic ORIs are sequestered on the nuclear matrix; this attachment is supposed to facilitate execution of their activation/deactivation programs during development. Furthermore, ORIs fall into various classes with respect to their sequence complexity: those enriched in AT tracts, those with GA- and CT-rich tracts, a smaller class of GC-rich ORIs, and a major class composed of mixed motifs yet containing distinct AT and polypurine or GC stretches. Multimers of an initiator protein in prokaryotes and viruses that might have evolved into a multiprotein replication initiation complex in multicellular organisms bind to the core ORI, causing a structural distortion to the DNA which is transferred to the AT tract flanking the initiator protein site; single-stranded DNA-binding proteins then interact with the melted AT tract as well as with the DNA polymerase alpha-primase complex in animal viruses and mammalian cells, causing initiation in DNA replication. ORIs in mammalian cells seem to colocalize with matrix-attached regions and are proposed to become DNase I-hypersensitive during their activation.

Animals↗

PubMed1996

Binding of MAR-DNA elements by mutant p53: possible implications for its oncogenic functions.

The tumor suppressor p53 is a multifunctional protein whose main duty is to preserve the integrity of the genome. This function of wild-type p53 as "guardian of the genome" is achieved at different levels, as a cell cycle checkpoint protein, halting the cell cycle upon DNA damage, and via a direct involvement in processes of DNA repair. Alternatively, p53 can induce apoptosis. Mutations in the p53 gene occur in about 50% of all human tumors and eliminate the tumor suppressor functions of p53. However, many mutant p53 proteins have not simply lost tumor suppressor functions but have gained oncogenic properties which contribute to the progression of tumor cells to a more malignant phenotype. The molecular basis for this gain of function of mutant p53 is still unknown. However, mutant (mut) p53 specifically binds to nuclear matrix attachment region (MAR) DNA elements. MAR elements constitute important higher order regulatory elements of chromatin structure and function. By binding to these elements, mut p53 could modulate important cellular processes, like gene expression, replication, and recombination, resulting in phenotypic alterations of the tumor cells. Mut p53 thus could be the first representative of a new class of oncogenes, which exert their functions via long-range alterations or perturbation of chromatin structure and function.

Animals↗

PubMed1999

Detection of DNA curvature by transverse pore gradient gel electrophoresis on PhastSystem gels.

It has been known since 1990 that DNA curvature can be recognized on transverse pore gradient gels by an intersection of "Ferguson curves" with those of DNA size standards. The miniaturized PhastSystem polyacrylamide gels allow one to detect DNA curvature effortlessly and fast and at great economy of sample relative to alternative methods of electrophoresis. Using the transverse gradient gel electrophoresis method, it was found that the 660 bp length subfragment of the matrix attachment region (MAR) sequence of the chicken lysosyme gene migrates as a fragment of 800-900 bp length. When subjected to digestion with the restriction enzyme HaeIII, the fragment gives rise to two species of 248 and 412 bp length, respectively. The Ferguson curves of both species intersect with those of DNA size standards, indicating that both exhibit curvature. Only the curvature of the 412 bp fragment conforms to prediction. Ethidium bromide abolishes the effect of curvature on the fragment, reducing its apparent size from 900 to 660, the value obtained by agarose gel electrophoresis.

Animals↗

PubMed2003

A hypothesis for chromatin domain opening.

The eukaryotic genome is organized into different domains by cis-acting elements, such as boundaries/insulators and matrix attachment regions, and is packaged with different degrees of condensation. In the M phase, the chromatin becomes further highly condensed into chromosomes. The first step for transcriptional activation of a given gene, at a particular time during development, in any locus, is the opening of its chromatin domain. This locus needs to be kept in this state in each early G(1) phase during every cell cycle. Certain distal enhance elements, including locus control regions (LCRs) and enhancers, are believed to perform this target chromatin domain opening process and several models have been proposed to explain distal enhance action. But they did not explain precisely how a given chromatin domain is opened. Based on various studies, we propose a hypothesis for the mechanism of opening chromatin on a large scale. One important mechanism may involved breaking one or two DNA strands and reducing the linking numbers within chromatin domain. The topological changes can overpass some complexes formed on DNA strands and can be transmitted from specific localized points over a broad region, until boundary elements or insulators are reached. These may initiate downstream events such as propagation of histone acetylation and the binding of transcription factors to proximal promoters and may further augment the action mediated by distal enhancer elements.

Chromatin↗

PubMed1995

Reciprocal homologous recombination in or near antibody VDJ genes.

To determine if rearranged heavy chain variable (VDJ) genes can recombine with each other by crossing over of DNA strands, we constructed a transgene that contained a promoter, VDJ gene, reporter gene to detect crossover events, intron enhancer, matrix attachment region, and constant gene for IgM (C mu). Following immunization of transgenic mice, hybrid molecules were isolated from B cell DNA which contained the transgene recombined with the endogenous IgH locus. Reciprocal products of crossovers were detected by plasmid rescue and PCR amplification, and they were sequenced. Recombination occurred somewhere within 147 bp of homology that contained the JH4 gene segment and 3' flanking DNA. The recombined transgenes had a 20-fold increase in mutation in the VDJ region compared to nonrecombined transgenes, which indicates that DNA sequences 3' of the C mu gene in the endogenous IgH locus are necessary for full activity of the mutator mechanism. The recovery of recombinants between VDJ transgenes and endogenous VDJ genes raises the possibility that reciprocal recombination may somatically diversify rearranged genes between maternal and paternal alleles.

Animals↗

PubMed1997

Transgenic expression of a CD46 (membrane cofactor protein) minigene: studies of xenotransplantation and measles virus infection.

CD46 (membrane cofactor protein) is a human cell-surface regulator of activated complement and a receptor for the measles virus. A CD46 transgenic mouse line with an expression pattern similar to that of human tissues has been produced, to develop an animal model of (i) the control of complement activation by complement regulators in hyperacute rejection of xenografts, and (ii) measles virus infection. The mouse line was made using a CD46 minigene that includes promoter sequence and the first two introns of genomic CD46, which was coinjected into mouse ova with chicken lysozyme matrix attachment region DNA. A high level of CD46 expression in homozygotic transgenic mice was obtained with spleen cells having approximately 75% of the level found on human peripheral blood mononuclear cells. CD46 was detected in all tissues examined by immunohistochemistry, radioimmunoassay and Western blotting, showing that these mice were suitable for transplantation and measles virus infection studies. It also indicated that the transgene included the important regulatory elements of the CD46 promoter. Transgenic spleen cells were significantly protected in vitro from human complement activated by either the classical or alternative pathways and from alternative pathway rat complement. Furthermore, transgenic mouse hearts transplanted to rats regulated complement deposition in an in vivo model of antibody-dependent hyperacute xenograft rejection. Similar to human lymphocytes, transgenic lymphoblasts could be infected in vitro with measles virus; infected cells expressed viral proteins and produced infectious viral particles. The data demonstrate the suitability of this minigene for obtaining high-level CD46 expression sufficient for enhanced resistance of transgenic cells to complement attack and for obtaining wide tissue distribution of CD46, analogous to human tissues and, therefore, useful for comparative studies.

Acute Disease↗

PubMed1993

Regulatory elements in the immunoglobulin kappa locus induce c-myc activation and the promoter shift in Burkitt's lymphoma cells.

In Burkitt's lymphoma cells the proto-oncogene c-myc is constantly juxtaposed through chromosomal translocation to one of the immunoglobulin loci on chromosomes 14, 2 or 22. In the majority of cases the chromosomal breakpoint is localized 3' or 5' of the gene leaving the physiological c-myc transcription unit intact. As a consequence of the translocation the c-myc gene on the translocation chromosome becomes transcriptionally activated in such a manner that the c-myc promoter P1 is more active than promoter P2. In order to define elements involved in c-myc activation through t(2;8) translocation we have studied the expression of constructs consisting of c-myc and different parts of the immunoglobulin kappa locus after stable transfection into Burkitt's lymphoma cells. The c-myc gene under the control of the complete Ig kappa locus containing matrix attachment region, intron enhancer, constant kappa gene and 3' enhancer was strongly activated with predominant usage of promoter P1. Deletion analysis revealed that the intron or 3' enhancers alone activated c-myc to a much lesser extent and with normal promoter usage (P1 < P2). The cooperation of the same regulatory elements is required not only for transcriptional activation and induction of the promoter shift but also for down-regulation of promoter P1 of the translocated c-myc allele by sodium butyrate, another characteristic feature of Burkitt's lymphoma cells. This supports the notion that all elements involved in transcriptional activation and dysregulation of c-myc are contained within the myc-Ig specific minichromosome.

Base Sequence↗

PubMed2005

A global but stable change in HeLa cell morphology induces reorganization of DNA structural loop domains within the cell nucleus.

DNA of higher eukaryotes is organized in supercoiled loops anchored to a nuclear matrix (NM). The DNA loops are attached to the NM by means of non-coding sequences known as matrix attachment regions (MARs). Attachments to the NM can be subdivided in transient and permanent, the second type is considered to represent the attachments that subdivide the genome into structural domains. As yet very little is known about the factors involved in modulating the MAR-NM interactions. It has been suggested that the cell is a vector field in which the linked cytoskeleton-nucleoskeleton may act as transducers of mechanical information. We have induced a stable change in the typical morphology of cultured HeLa cells, by chronic exposure of the cells to the polar compound dimethylsulfoxide (DMSO). Using a PCR-based method for mapping the position of any DNA sequence relative to the NM, we have monitored the position relative to the NM of sequences corresponding to four independent genetic loci located in separate chromosomes representing different territories within the cell nucleus. Here, we show that stable modification of the NM morphology correlates with the redefinition of DNA loop structural domains as evidenced by the shift of position relative to the NM of the c-myc locus and the multigene locus PRM1 --> PRM2 --> TNP2, suggesting that both cell and nuclear shape may act as cues in the choice of the potential MARs that should be attached to the NM.

Cell Nucleus↗

PubMed2002

A major breakpoint cluster domain in murine radiation-induced acute myeloid leukemia.

Cytogenetic and molecular studies have provided evidence of the clustering of chromosome 2 deletion breakpoints in radiation-induced murine acute myeloid leukemia (AML). Moreover, clustering occurs in at least two fragile domains rich in telomere-like arrays. Here we describe a physical map of the distal breakpoint cluster and confirm the presence of inverted head-to-head telomeric sequence arrays. These potentially recombinogenic sequences were not, however, the direct focus for post-irradiation chromosome breakage in AML. Instead, the two arrays bordered a 2.5-kb sequence with properties expected of a nuclear matrix attachment region (MAR). The putative MAR co-localized in the fragile domain with genes important to the hemopoietic system (leukocyte tyrosine kinase, zinc finger protein 106, erythrocyte protein band 4.2, and beta(2)-microglobulin (beta2m)); the beta2m subdomain was a particular focus of breakage. On the basis of these and other data, we suggest that AML-associated chromosome 2 fragility in the mouse is a consequence of domain-specific fragility in genomic domains containing numerous genes critical to the hemopoietic system. Recorded with the permission of the controller of Her Majesty's Stationery Office. Published by Wiley-Liss, Inc.

Acute Disease↗

PubMed1997

Ligation-mediated PCR for chromatin-structure analysis of interphase and metaphase chromatin.

Chromatin structure is becoming increasingly recognized as important for a full understanding of gene function and cell memory. With regard to cell memory, which involves the transfer of chromatin-encoded epigenetic information from one cell generation to another, the detailed structure of metaphase chromatin is of crucial importance. In this paper we describe methods for the use of dimethyl sulfate, DNase I, and potassium permanganate for in vivo footprinting and chromatin analysis, with special emphasis on studies of metaphase cells. We review the use of ligation-mediated PCR for the analysis of chromatin, including the human phosphoglycerate kinase promoter, and also report initial studies of a matrix attachment region near the human beta-interferon gene.

Cell Line↗