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PubMed2002

Molecular properties of a matrix attachment region-binding protein located in the nucleoli of tobacco cells.

We cloned a cDNA for matrix-attachment region (MAR)-binding protein from Nicotiana tabacum cells to elucidate the structure and function of the nuclear matrix. The cDNA encodes a protein of 555 amino acids (61,050 Da) with an isoelectric point of 9.4. We named the protein NtMARBP61. The sequence is 45% identical to yeast Nop58p, which is involved in rRNA processing. The C-terminal part is unique and rich in lysine residues. The recombinant C-terminal part had the ability to bind double-stranded DNAs of 12 tobacco MARs. The intracellular localization was determined to be in the nucleolus by fluorescent microscopy using the antibody to the recombinant NtMARBP61. The mRNA level was high in the lag and early-log phases of cultured cells but low in the stationary phase. The protein was accumulated only in the middle- and late-log phases, suggesting that NtMARBP61 is essential for growing cells. The results suggest at least the structural and regulatory function of NtMARBP61 in the nucleolus as a MAR-binding protein in a growth-stage specific manner.

Amino Acid Sequence↗

PubMed2000

Conservation of matrix attachment region-binding filament-like protein 1 among higher plants.

The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) on the DNA is considered to be of fundamental importance for higher-order chromatin organization and the regulation of gene expression. We have previously isolated a novel nuclear matrix-localized protein (MFP1) from tomato (Lycopersicon esculentum) that preferentially binds to MAR DNA. Tomato MFP1 has a predicted filament-protein-like structure and is associated with the nuclear envelope via an N-terminal targeting domain. Based on the antigenic relationship, we report here that MFP1 is conserved in a large number of dicot and monocot species. Several cDNAs were cloned from tobacco (Nicotiana tabacum) and shown to correspond to two tobacco MFP1 genes. Comparison of the primary and predicted secondary structures of MFP1 from tomato, tobacco, and Arabidopsis indicates a high degree of conservation of the N-terminal targeting domain, the overall putative coiled-coil structure of the protein, and the C-terminal DNA-binding domain. In addition, we show that tobacco MFP1 is regulated in an organ-specific and developmental fashion, and that this regulation occurs at the level of transcription or RNA stability.

Amino Acid Sequence↗

PubMed2003

Amplification of plasmids containing a mammalian replication initiation region is mediated by controllable conflict between replication and transcription.

We previously showed that plasmids containing both a mammalian replication initiation region and a matrix attachment region were efficiently amplified in human cancer cells and that they were either integrated into preexisting extrachromosomal double minutes (DMs) or induced the generation of a chromosomal homogeneously staining region (HSR). In this article, we elucidated the mechanism by which such plasmids mimic gene amplification. Hybridization experiments using chromatin fiber, metaphase spread, and genomic Southern blot analysis suggested that a circular molecule comprising a plasmid direct repeat was generated initially. Recombination between this molecule and the preexisting DMs led to the apparent stabilization of the plasmid repeat. If the plasmid repeat was integrated into the chromosome, it initiated the breakage-fusion-bridge cycle, which generated HSR. Importantly, we found that HSR formation was blocked by inserting a poly(A) signal or the orientation-specific replication fork barrier downstream of the drug-resistance gene, where the transcription would meet head to head with the supposed replication fork from the initiation region. The matrix attachment region enhanced HSR formation if it was inserted at the same site. These data suggested that strand breakage generated by the conflict between replication and transcription might trigger the breakage-fusion-bridge cycle. This is the first study suggesting that such a conflict leads to genomic instability in higher eukaryotes.

Animals↗

PubMed2000

Regulation of matrix attachment region-dependent, lymphocyte-restricted transcription through differential localization within promyelocytic leukemia nuclear bodies.

Bright (B cell regulator of IgH transcription) transactivates the immunoglobulin heavy chain (IgH) intronic enhancer, Emicro, by binding to matrix attachment regions (MARs), sites necessary for DNA attachment to the nuclear matrix. Here we report that Bright interacts with the ubiquitous autoantigen Sp100, a component of promyelocytic leukemia nuclear bodies (PML NBs), and with LYSp100B/Sp140, the lymphoid-restricted homolog of Sp100. Both in intact cells and in nuclear matrix preparations, the majority of Bright and Sp100 colocalize within PML NBs. In contrast, Bright colocalizes with only a small fraction of LYSp100B while inducing a redistribution of the majority of LYSp100B from its associated nuclear domains (LANDs) into nucleoplasm and cytoplasm. Sp100 represses the MAR-binding and transactivation activity of Bright. LYSp100B interacts more weakly with Bright but requires significantly higher levels than Sp100 to inhibit MAR binding. However, it strongly stimulates Bright transactivation through E mu. We suggest that Sp100 and LYSp100B interactions with Bright have different consequences for IgH transcription, potentially through differential association of E mu MARs with nuclear matrix- associated PML NBs and LANDs.

Antigens, Nuclear↗

PubMed1997

A thymocyte factor SATB1 suppresses transcription of stably integrated matrix-attachment region-linked reporter genes.

SATB1 specifically recognizes and binds to specialized genomic regions with an ATC sequence context with high base-unpairing propensity. Such base-unpairing regions (BURs) are typically identified within nuclear scaffold- or matrix-attachment regions (S/MARs). SATB1 is a homeodomain protein and is predominantly expressed in thymocytes. We obtained BHK cell lines expressing low levels of SATB1 by stable transfection and investigated its effect on stably integrated MAR-linked SV40 enhancer/promoter-driven luciferase reporter genes. For this study, both naturally occurring and synthetic MARs, as well as an AT-rich non-MAR control, were tested. Previous studies demonstrated that MAR sequences augment transcription of the linked reporter luciferase gene. Here, we show that SATB1 dramatically reduces the high levels of MAR-linked luciferase gene transcription. Transcription was virtually abolished for a reporter gene surrounded by two MARs at the 5' and 3' ends of the gene, which otherwise confer the highest level of transcriptional augmentation. On the other hand, SATB1 did not affect expression of an AT-rich non-MAR-linked luciferase gene or of endogenous housekeeping genes. This study shows that SATB1 acts as a strong transcriptional suppressor on a reporter gene linked to MARs when it is stably integrated into chromatin.

Animals↗

PubMed2005

Solution structure and DNA-binding mode of the matrix attachment region-binding domain of the transcription factor SATB1 that regulates the T-cell maturation.

SATB1 is a transcriptional regulator controlling the gene expression that is essential in the maturation of the immune T-cell. SATB1 binds to the nuclear matrix attachment regions of DNA, where it recruits histone deacetylase and represses transcription through a local chromatin remodeling. Here we determined the solution structure of the matrix attachment region-binding domain, possessing similarity to the CUT DNA-binding domain, of human SATB1 by NMR spectroscopy. The structure consists of five alpha-helices, in which the N-terminal four are arranged similarly to the four-helix structure of the CUT domain of hepatocyte nuclear factor 6alpha. By an NMR chemical shift perturbation analysis and by surface plasmon resonance analyses of SATB1 mutant proteins, an interface for DNA binding was revealed to be located at the third helix and the surrounding regions. Surface plasmon resonance experiments using groove-specific binding drugs and methylated DNAs indicated that the domain recognizes DNA from the major groove side. These observations suggested that SATB1 possesses a DNA-binding mode similar to that of the POU-specific DNA-binding domain, which is known to share structural similarity to the four-helix CUT domain.

Amino Acid Sequence↗

PubMed1997

The matrix attachment region-binding protein SATB1 participates in negative regulation of tissue-specific gene expression.

The nuclear matrix has been implicated in several cellular processes, including DNA replication, transcription, and RNA processing. In particular, transcriptional regulation is believed to be accomplished by binding of chromatin loops to the nuclear matrix and by the concentration of specific transcription factors near these matrix attachment regions (MARs). A number of MAR-binding proteins have been identified, but few have been directly linked to tissue-specific transcription. Recently, we have identified two cellular protein complexes (NBP and UBP) that bind to a region of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) previously shown to contain at least two negative regulatory elements (NREs) termed the promoter-proximal and promoter-distal NREs. These NREs are absent from MMTV strains that cause T-cell lymphomas instead of mammary carcinomas. We show here that NBP binds to a 22-bp sequence containing an imperfect inverted repeat in the promoter-proximal NRE. Previous data showed that a mutation (p924) within the inverted repeat elevated basal transcription from the MMTV promoter and destabilized the binding of NBP, but not UBP, to the proximal NRE. By using conventional and affinity methods to purify NBP from rat thymic nuclear extracts, we obtained a single major protein of 115 kDa that was identified by protease digestion and partial sequencing analysis as the nuclear matrix-binding protein special AT-rich sequence-binding protein 1 (SATB1). Antibody ablation, distamycin inhibition of binding, renaturation and competition experiments, and tissue distribution data all confirmed that the NBP complex contained SATB1. Similar types of experiments were used to show that the UBP complex contained the homeodomain protein Cux/CDP that binds the MAR of the intronic heavy-chain immunoglobulin enhancer. By using the p924 mutation within the MMTV LTR upstream of the chloramphenicol acetyltransferase gene, we generated two strains of transgenic mice that had a dramatic elevation of reporter gene expression in lymphoid tissues compared with reporter gene expression in mice expressing wild-type LTR constructs. Thus, the 924 mutation in the SATB1-binding site dramatically elevated MMTV transcription in lymphoid tissues. These results and the ability of the proximal NRE in the MMTV LTR to bind to the nuclear matrix clearly demonstrate the role of MAR-binding proteins in tissue-specific gene regulation and in MMTV-induced oncogenesis.

Amino Acid Sequence↗

PubMed2003

Nucleic acid-matrix attachment recognition regions--as facilitators in plasmid transfer.

Non-viral gene transfer is an alternative to viral vectors for gene transfer. However, non-viral transgene expression remains undesirably low and transient. Matrix attachment regions (MARs) are DNA elements that are defined by their high affinity for the nuclear matrix. MARs may also be related to long-term transgene expression in vitro. The purpose of this research is to evaluate human interferon-beta MARs element in various cell types. This was accomplished by constructing MARs-containing plasmid DNA (pDNA) and comparing their transgene expression with non-MARs-containing pDNA. We found that MARs-containing pDNA increased and prolonged the expression in Chinese hamster ovary (CHO) cells, but not in human neuroblastoma cells (SKnSH) and neuronal cells (primary neuron, astroglia and microglia). From the cotransfection experiment, MARs-containing pDNA had a trans effect on another pDNA expression. A PCR method was used to monitor the intracellular distribution of pDNA after cellular fractionalization. We found that non-MAR containing pDNA demonstrates similar intracellular distribution as non-MARs-containing pDNA.

Animals↗

PubMed1990

Drosophila scaffold-attached regions bind nuclear scaffolds and can function as ARS elements in both budding and fission yeasts.

Histone-depleted nuclei maintain sequence-specific interactions with genomic DNA at sites known as scaffold attachment regions (SARs) or matrix attachment regions. We have previously shown that in Saccharomyces cerevisiae, autonomously replicating sequence elements bind the nuclear scaffold. Here, we extend these observations to the fission yeast Schizosaccharomyces pombe. In addition, we show that four SARs previously mapped in the genomic DNA of Drosophila melanogaster bind in vitro to nuclear scaffolds from both yeast species. In view of these results, we have assayed the ability of the Drosophila SARs to promote autonomous replication of plasmids in the two yeast species. Two of the Drosophila SARs have autonomously replicating sequence activity in budding yeast, and three function in fission yeast, while four flanking non-SAR sequences are totally inactive in both.

Animals↗

PubMed1997

Mathematical model to predict regions of chromatin attachment to the nuclear matrix.

The potentiation and subsequent initiation of transcription are complex biological phenomena. The region of attachment of the chromatin fiber to the nuclear matrix, known as the matrix attachment region or scaffold attachment region (MAR or SAR), are thought to be requisite for the transcriptional regulation of the eukaryotic genome. As expressed sequences should be contained in these regions, it becomes significant to answer the following question: can these regions be identified from the primary sequence data alone and subsequently used as markers for expressed sequences? This paper represents an effort toward achieving this goal and describes a mathematical model for the detection of MARs. The location of matrix associated regions has been linked to a variety of sequence patterns. Consequently, a list of these patterns is compiled and represented as a set of decision rules using an AND-OR formulation. The DNA sequence was then searched for the presence of these patterns and a statistical significance was associated with the frequency of occurrence of the various patterns. Subsequently, a mathematical potential value,MAR-Potential, was assigned to a sequence region as the inverse proportion to the probability that the observed pattern population occurred at random. Such a MAR detection process was applied to the analysis of a variety of known MAR containing sequences. Regions of matrix association predicted by the software essentially correspond to those determined experimentally. The human T-cell receptor and the DNA sequence from the Drosophila bithorax region were also analyzed. This demonstrates the usefulness of the approach described as a means to direct experimental resources.

Base Sequence↗

PubMed1999

HMG-I(Y) recognizes base-unpairing regions of matrix attachment sequences and its increased expression is directly linked to metastatic breast cancer phenotype.

Base-unpairing regions (BURs) contain a specialized DNA context with an exceptionally high unwinding propensity, and are typically identified within various matrix attachment regions. A BUR affinity column was used to purify a doublet of Mr 20,000 proteins from human breast carcinoma cells. These proteins were identified as the high-mobility group (HMG) protein, HMG-I, and its splicing variant, HMG-Y. We show that HMG-I(Y) specifically binds BURs. Mutating BURs so as to abrogate their unwinding property greatly reduced their binding affinity to HMG-I(Y). Numerous studies have indicated that elevated HMG-I(Y) expression is correlated with more advanced cancers and with increased metastatic potential. We studied whether the expression of HMG-I(Y) responds to signaling through the heregulin (HRG)-erbB pathway and the extracellular matrix. HMG-I(Y) expression was increased in MCF-7 cells after stable transfection with an HRG expression construct that led cells to acquire estrogen independence and metastasizing ability. A high level of HMG-I(Y) expression was detected in metastatic MDA-MB-231 cells, but the expression was virtually diminished, and the metastasizing ability was lost after cells were stably transfected with an antisense HRG cDNA construct. HMG-I(Y) was also decreased in MDA-MB-231 cells when treated with a chemical inhibitor for matrix metalloproteinase-9 that led to a reduction of invasive capability in vitro. The level of HMG-I(Y) expression, therefore, is dynamically regulated in human breast cancer cells in response to varying types of signaling that affect metastatic ability, including the HRG-erbB pathway and those from the extracellular matrix.

Antisense Elements (Genetics)↗

PubMed2001

Matrix attachment region-dependent function of the immunoglobulin mu enhancer involves histone acetylation at a distance without changes in enhancer occupancy.

Nuclear matrix attachment regions (MARs), which flank the immunoglobulin mu heavy-chain enhancer on either side, are required for the activation of the distal variable-region (V(H)) promoter in transgenic mice. Previously, we have shown that the MARs extend a local domain of chromatin accessibility at the mu enhancer to more distal sites. In this report, we examine the influence of MARs on the formation of a nucleoprotein complex at the enhancer and on the acetylation of histones, which have both been implicated in contributing to chromatin accessibility. By in vivo footprint analysis of transgenic mu gene constructs, we show that the occupancy of factor-binding sites at the mu enhancer is similar in transcriptionally active wild-type and transcriptionally inactive DeltaMAR genes. Chromatin immunoprecipitation experiments indicate, however, that the acetylation of histones at enhancer-distal nucleosomes is enhanced 10-fold in the presence of MARs, whereas the levels of histone acetylation at enhancer-proximal nucleosomes are similar for wild-type and DeltaMAR genes. Taken together, these data indicate that the function of MARs in mediating long-range chromatin accessibility and transcriptional activation of the V(H) promoter involves the generation of an extended domain of histone acetylation, independent of changes in the occupancy of the mu enhancer.

Acetylation↗

PubMed1996

The human clotting factor VIII cDNA contains an autonomously replicating sequence consensus- and matrix attachment region-like sequence that binds a nuclear factor, represses heterologous gene expression, and mediates the transcriptional effects of sodium butyrate.

Expression of the human blood-clotting factor VIII (FVIII) cDNA is hampered by the presence of sequences located in the coding region that repress transcription. We have previously identified a 305-bp fragment within the FVIII cDNA that is involved in the repression (R.C. Hoeben, F.J. Fallaux, S.J. Cramer, D.J.M. van den Wollenberg, H. van Ormondt, E. Briet, and A.J. van der Eb, Blood 85:2447-2454, 1995). Here, we show that this 305-bp region of FVIII cDNA contains sequences that resemble the yeast (Saccharomyces cerevisiae) autonomously replicating sequence consensus. Two of these DNA elements coincide with AT-rich sequences that are often found in matrix attachment regions or scaffold-attached regions. One of these elements, consisting of nucleotides 1569 to 1600 of the FVIII cDNA (nucleotide numbering is according to the system of Wood et al. (W.I. Wood, D.J. Capon, C.C. Simonsen, D.L. Eaton, J. Gitschier, D. Keyt, P.H. Seeburg, D.H. Smith, P. Hollingshead, K.L. Wion, et al., Nature [London] 312:330-337,1984), binds a nuclear factor in vitro but loses this capacity after four of its base pairs have been changed. A synthetic heptamer of this segment can repress the expression of a chloramphenicol acetyltransferase (CAT) reporter gene and also loses this capacity upon mutation. Furthermore, we demonstrate that repression by FVIII sequences can be relieved by sodium butyrate. We demonstrate that the synthetic heptamer (FVIII nucleotides 1569 to 1600), when placed upstream of the Moloney murine leukemia virus long terminal repeat promoter that drives the CAT reporter, can render the CAT reporter inducible by butyrate. This effect was absent when the same element was mutated. The stimulatory effect of butyrate could not be attributed to butyrate-responsive elements in the studied long terminal repeat promoters. Our data provide a functional characterization of the sequences that repress expression of the FVIII cDNA. These data also suggest a link between transcriptional repression by FVIII cDNA elements and the stimulatory effect of butyrate on FVIII cDNA expression.

Base Sequence↗

PubMed1992

Sequence-specific DNA-binding proteins are components of a nuclear matrix-attachment site.

We have identified a nuclear matrix-attachment region within an upstream element of a human H4 histone gene promoter. Nuclear matrix proteins, isolated and solubilized from HeLa S3 cells, were found to interact with sequence specificity at this matrix-attachment region. Several types of assays for protein-DNA interaction showed that the minimal sequence for the nuclear matrix protein-DNA interaction was 5'-TGACGTCCATG-3'; the underlined region corresponds to the core consensus sequence for ATF transcription factor binding. Two proteins with molecular masses of 43 and 54 kDa were identified by UV-crosslinking analysis as integral components of this protein-DNA complex. The molecular masses of these proteins and the ATF-binding site consensus sequence suggest that these proteins are members of the ATF family. Our results provide direct evidence for nuclear matrix localization of sequence-specific DNA-binding factors for an actively transcribed gene. The proximity of a strong positive transcriptional regulatory element to the matrix-attachment region of this gene suggests that the nuclear matrix may serve to localize and concentrate trans-acting factors that facilitate regulation of gene expression.

Base Sequence↗

PubMedDate not supplied

Agrobacterium-mediated transformation of herbicide resistance in creeping bentgrass and colonial bentgrass.

Embryogenic calli were induced from the seeds of creeping bentgrass (Agrostis palustris Huds.) cv. Regent and colonial bentgrass (Agrostis Tenuis Sibth. Fl. Oxen.) cv. Tiger. The embryogenic calli were precultured on fresh medium for 4-7 days and then co-cultivated with Agrobacterium tumefaciens, LBA4404, which contains plasmid vector-pSBGM harboring bar coding region, synthetic green fluorescent protein (sGFP) coding region and matrix attachment region (MAR). After 3 days of co-cultivation, the calli were washed thoroughly and transferred to MS medium containing 2 mg/L of 2, 4-D, 12-15 mg/L phosphinothricin (PPT) and 250 mg/L of cefotaxime. After 2-3 months of selection, the actively growing calli of 'Regent' and 'Tiger' were transferred to MS medium with 12-15 mg/L PPT and 250 mg/L cefotaxime for regeneration. The putative transformants were maintained on MS medium with 3 mg/L PPT for long period but control died within 1 month. After establishing in greenhouse, the transformants also showed strong resistance to 0.4% of herbicide Basta but control plants died within 2 weeks. Under confocal microscope, both young leaves and roots showed significant GFP expression. PCR analysis revealed the presence of a DNA fragment of GFP gene at the expected size (380 bp) in the transformants and its absence in a randomly selected control plant.

Agrostis↗

PubMed1993

Association of putative origins of replication with the nuclear matrix in normal human fibroblasts.

Several metabolic processes, such as DNA organization and replication, transcription, and RNA processing are closely associated with the nuclear matrix. Nuclear matrix attachment regions are nucleotide sequences holding DNA tightly complexed with the nuclear scaffold and are resistant to extractions with detergents and high salt solutions. The role of matrix attachment regions in DNA replication has not been completely clarified, but they have been identified in close association with origins of replication in mammalian cells. We isolated nuclear matrix-associated DNA from normal human fibroblasts synchronized to different phases of the cell cycle and cloned compatible fragments into pUC19. We tested the homology of a fraction of the available clones to DNA replicated at the beginning of the S phase in human fibroblasts. We confirmed that nuclear matrix-associated DNA isolated from cells in G0 and G1 phases of the cell cycle contains sequences that are among the earliest replicated regions in the human genome. This finding supports the hypothesis that matrix attachment regions in human DNA are located in close proximity to origins of DNA replication.

Cells, Cultured↗

PubMed2001

The matrix attachment region-binding protein SATB1 interacts with multiple elements within the gp91phox promoter and is down-regulated during myeloid differentiation.

The gp91(phox) gene encodes a component of the respiratory burst NADPH oxidase complex and is highly expressed in mature myeloid cells. The transcriptional repressor CCAAT displacement protein binds to at least five sites within the proximal gp91(phox) promoter and represses expression prior to terminal phagocyte differentiation. The DNA binding activity of CCAAT displacement protein decreases during terminal phagocyte differentiation, thus permitting the binding of transcriptional activators and induction of gp91(phox) expression. We report here that the matrix attachment region-binding protein SATB1 interacts with at least seven sites within the -1542 to +12-base pair gp91(phox) promoter. Four additional binding sites for CCAAT displacement protein were also identified. Furthermore, the most proximal SATB1-binding site within the gp91(phox) promoter binds specifically to the nuclear matrix fraction in vitro. SATB1 expression is down-regulated during terminal myeloid cell differentiation, coincident with induction of gp91(phox) expression. Transient transfection assays demonstrate that a SATB1-binding site derived from the gp91(phox) promoter represses promoter activity in cells expressing SATB1. These findings underscore the importance of transcriptional repression in the regulation of gp91(phox) expression and reveal a candidate myeloid cell target gene for SATB1, a factor previously found to be essential for T cell development.

Animals↗

PubMed1998

The 5'-flanking cis-acting elements of the human epsilon-globin gene associates with the nuclear matrix and binds to the nuclear matrix proteins.

The nuclear matrix attachment regions(MARs) and the binding nuclear matrix proteins in the 5'-flanking cis-acting elements of the human epsilon-globin gene have been examined. Using in vitro DNA-matrix binding assay, it has been shown that the positive stage-specific regulatory element (epsilon-PREII, -446 bp(-)-419 bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells, indicating that epsilon-PREII may be an erythroid-specific facultative MAR. In gel mobility shift assay and Southwestern blotting assay, an erythroid-specific nuclear matrix protein (epsilon-NMP kappa) in K562 cells has been revealed to bind to this positive regulatory element (epsilon-PREII). Furthermore, we demonstrated that the silencer (-392 bp(-)-177 bp) upstream of the human epsilon-globin gene could associate with the nuclear matrices from K562, HEL and Raji cells. In addition, the nuclear matrix proteins prepared from these three cell lines could also bind to this silencer, suggesting that this silencer element might be a constitutive nuclear matrix attachment region (constitutive MAR). Our results demonstrated that the nuclear matrix and nuclear matrix proteins might play an important role in the regulation of the human epsilon-globin gene expression.

Antigens, Nuclear↗