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Characterization of SAF-A, a novel nuclear DNA binding protein from HeLa cells with high affinity for nuclear matrix/scaffold attachment DNA elements. 总被引:6,自引:0,他引:6
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We identified four proteins in nuclear extracts from HeLa cells which specifically bind to a scaffold attachment region (SAR) element from the human genome. Of these four proteins, SAF-A (scaffold attachment factor A), shows the highest affinity for several homologous and heterologous SAR elements from vertebrate cells. SAF-A is an abundant nuclear protein and a constituent of the nuclear matrix and scaffold. The homogeneously purified protein is a novel double stranded DNA binding protein with an apparent molecular weight of 120 kDa. SAF-A binds at multiple sites to the human SAR element; competition studies with synthetic polynucleotides indicate that these sites most probably reside in the multitude of A/T-stretches which are distributed throughout this element. In addition we show by electron microscopy that the protein forms large aggregates and mediates the formation of looped DNA structures. 相似文献
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A matrix/scaffold attachment region binding protein: identification, purification, and mode of binding. 总被引:32,自引:0,他引:32
Matrix/scaffold attachment regions (MARs/SARs) partition chromatin into functional loop domains. Here we have identified a chicken protein that selectively binds to MARs from the chicken lysozyme locus and to MARs from Drosophila, mouse, and human genes. This protein, named ARBP (for attachment region binding protein), was purified to homogeneity and shown to bind to MARs in a cooperative fashion. ARBP is an abundant nuclear protein and a component of the internal nuclear network. Deletion mutants indicate that multiple AT-rich sequences, if contained in a minimal approximately 350 bp MAR fragment, can lead to efficient binding of ARBP. Furthermore, dimerization mutants show that, to bind ARBP efficiently, MAR sequences can act synergistically over large distances, apparently with the intervening DNA looping out. The binding characteristics of ARBP to MARs reproduce those of unfractionated matrix preparations, suggesting that ARBP is an important nuclear element for the generation of functional chromatin loops. 相似文献
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Traweger A Fuchs R Krizbai IA Weiger TM Bauer HC Bauer H 《The Journal of biological chemistry》2003,278(4):2692-2700
Zonula occludens proteins (ZOPs), currently comprising ZO-1, ZO-2, and ZO-3, belong to the family of membrane-associated guanylate kinase homologue (MAGUK) proteins that are involved in the organization of epithelial and endothelial intercellular junctions. ZOPs bind to the cytoplasmic C termini of junctional transmembrane proteins linking them to the actin cytoskeleton. They are characterized by several conserved modules, including three PDZ domains, one SH3 domain, and a guanylate kinase-like domain, elements indicating that ZOPs may serve multiple purposes. Interestingly, ZOPs contain some unique motifs not shared by other MAGUK family members, including nuclear localization and nuclear export signals and a leucine zipper-like sequence. Their potential involvement in cell growth and proliferation has been suggested earlier based on the observation that the N-terminal half of ZOPs displays significant similarity to the product of the Drosophila tumor suppressor gene lethal(1)discs-large (dlg). The nuclear targeting of ZOPs in subconfluent epithelial cell cultures is well documented, although the action of the junctional MAGUKs in the nucleus has remained elusive. Here we show for the first time that nuclear ZO-2 directly interacts with the DNA-binding protein scaffold attachment factor-B (SAF-B). Our results from two-hybrid assays and in vivo co-immunoprecipitation studies provide evidence to suggest that ZO-2 associates with the C-terminal portion of SAF-B via its PDZ-1 domain. We further demonstrate that enhanced green fluorescent protein (EGFP)- and DsRed-tagged ZO-2 and SAF-B fusion proteins partially co-localize in nuclei of transfected epithelial cells. As shown by laser confocal microscopy and epifluorescent analysis, nuclear ZO-2 is present in epithelial and endothelial cells, particularly in response to environmental stress conditions. Interestingly, no association of SAF-B with ZO-1 was found, which supports the notion that junctional MAGUKs serve nonredundant functions. 相似文献
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One class of heterogeneous nuclear ribonucleoproteins (hnRNPs), AUF1/hnRNP D, consists of four isoform proteins (p45, p42, p40, and p37) which are generated by alternative splicing. The present study was therefore undertaken to clarify any isoform-specific differences in terms of their functions and nucleocytoplasmic localization. All isoforms primarily localized in the nucleus. However, heterokaryon analysis and a study using RNA polymerase II inhibitor revealed that p40/p37 exhibited a continuous shuttling between the nucleus and cytoplasm. Constant nuclear retention activity was mapped to the p45/p42-specific sequence at the C-terminal region, which is retained by alternative splicing. Using this domain as a probe, we performed a yeast two-hybrid screening and we found that scaffold attachment factor B (SAF-B), a nuclear matrix-associated protein, exhibits protein-protein interaction to this region. Colocalization of p45/p42 and SAF-B was observed as a speckle in the nucleus. Interestingly, p45/p42 isoforms appeared to act as a negative regulator in gene expression by forming a complex with SAF-B. Thus, the present study revealed that the isoform-specific functions of AUF1/hnRNP D are defined by intracellular shuttling capacity. 相似文献
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SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA
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Kipp M Göhring F Ostendorp T van Drunen CM van Driel R Przybylski M Fackelmayer FO 《Molecular and cellular biology》2000,20(20):7480-7489
SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix. The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood. We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins. This domain was first identified in human scaffold attachment factor A (SAF-A) and was thus designated SAF-Box. The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain. We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity. Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments. This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold. 相似文献
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Hideki Ogino Michihiko Fujii Wataru Satou Toshikazu Suzuki Eriko Michishita Dai Ayusawa 《DNA research》2002,9(1):25-29
Substitution of thymine with 5-bromouracil in DNA is known to change interaction between DNA and proteins, thereby inducing various biological phenomena. We hypothesize that A/T-rich scaffold/nuclear matrix attachment region (S/MAR) sequences are involved in the effects of 5-bromodeoxyuridine. We examined an interaction between DNA containing an intronic S/MAR sequence of the immunoglobulin heavy chain gene and nuclear halos prepared from HeLa cells. Upon substitution with 5-bromouracil, the S/MAR DNA bound more tightly to the nuclear halos. The multi-functional nuclear matrix protein YY1 was also found to bind more strongly to 5-bromouracil-substituted DNA containing its recognition motif. These results are consistent with the above hypothesis. 相似文献
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Attachment of origins of replication to the nuclear matrix and the chromosomal scaffold 总被引:7,自引:0,他引:7
We have investigated the attachment of DNA to the nuclear matrix and chromosomal scaffold in synchronized bovine liver cells. Label incorporated at the onset of the S phase remained preferentially associated with the matrix during the subsequent G1 phase and with a residual protein structure from dehistonized chromosomes during mitosis. On the other hand label incorporated during mid or late S phase was about equally distributed over the DNA molecule after a chase into the G1 phase. These results suggest that DNA is attached to the nuclear matrix and chromosome scaffolds by the origins of replication. 相似文献
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DNA loop organization by nuclear scaffold/matrix attachment is a key regulator of gene expression that may provide a means to modulate phenotype. We have previously shown that attachment of genes to the NaCl-isolated nuclear matrix correlates with their silencing in HeLa cells. In contrast, expressed genes were associated with the lithium 3,5-diiodosalicylate (LIS)-isolated nuclear scaffold. To define their role in determining phenotype matrix attached regions (MARs) on human chromosomes 14–18 were identified as a function of expression in a primary cell line. The locations of MARs in aortic adventitial fibroblast (AoAF) cells were very stable (r = 0.909) and 96% of genes attached at MARs are silent (P < 0.001). Approximately one-third of the genes uniquely expressed in AoAF cells were associated with the HeLa cell nuclear matrix and silenced. Comparatively, 81% were associated with the AoAF cell nuclear scaffold (P < 0.001) and expressed. This suggests that nuclear scaffold/matrix association mediates a portion of cell type-specific gene expression thereby modulating phenotype. Interestingly, nuclear matrix attachment and thus silencing of specific genes that regulate proliferation and maintain the integrity of the HeLa cell genome suggests that transformation may at least in part be achieved through aberrant nuclear matrix attachment. 相似文献
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DNA-PK-dependent binding of DNA ends to plasmids containing nuclear matrix attachment region DNA sequences: evidence for assembly of a repair complex 总被引:2,自引:0,他引:2
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We find that nuclear protein extracts from mammalian cells contain an activity that allows DNA ends to associate with circular pUC18 plasmid DNA. This activity requires the catalytic subunit of DNA-PK (DNA-PKcs) and Ku since it was not observed in mutants lacking Ku or DNA-PKcs but was observed when purified Ku/DNA-PKcs was added to these mutant extracts. Purified Ku/DNA-PKcs alone did not produce association of DNA ends with plasmid DNA suggesting that additional factors in the nuclear extract are necessary for this activity. Competition experiments between pUC18 and pUC18 plasmids containing various nuclear matrix attachment region (MAR) sequences suggest that DNA ends preferentially associate with plasmids containing MAR DNA sequences. At a 1:5 mass ratio of MAR to pUC18, approximately equal amounts of DNA end binding to the two plasmids were observed, while at a 1:1 ratio no pUC18 end binding was observed. Calculation of relative binding activities indicates that DNA end-binding activities to MAR sequences was 7–21-fold higher than pUC18. Western analysis of proteins bound to pUC18 and MAR plasmids indicates that XRCC4, DNA ligase IV and scaffold attachment factor A preferentially associate with the MAR plasmid in the absence or presence of DNA ends. In contrast, Ku and DNA-PKcs were found on the MAR plasmid only in the presence of DNA ends suggesting that binding of these proteins to DNA ends is necessary for their association with MAR DNA. The ability of DNA-PKcs/Ku to direct DNA ends to MAR and pUC18 plasmid DNA is a new activity for DNA-PK and may be important for its function in double-strand break repair. A model for DNA repair based on these observations is presented. 相似文献
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Sergey V. Razin Irina I. Gromova 《BioEssays : news and reviews in molecular, cellular and developmental biology》1995,17(5):443-450
The specificity of eukaryotic DNA organization into loops fixed to the nuclear matrix/chromosomal scaffold has been studied for more than fifteen years. The results and conclusions of different authors remain, however, controversial. Recently, we have elaborated a new approach to the study of chromosomal DNA loops. Instead of characterizing loop basements (nuclear matrix DNA), we have concentrated our efforts on the characterization of individual loops after their excision by DNA topoisomerase II-mediated DNA cleavage at matrix attachment sites. In this review the results of applying this mapping approach are compared with the results and conclusions from studies of nuclear matrix DNA. An attempt is also made to reconsider all data about the specificity of DNA interactions with the nuclear matrix and to suggest a model of spatial organization of the eukaryotic genome which resolves apparent contradictions between these data. 相似文献
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A. Ferraro L. Cervoni M. Eufemi F. Altieri C. Turano 《Journal of cellular biochemistry》1996,62(4):495-505
DNA-protein cross-linkages were formed in intact nuclei of chicken erythrocytes and liver cells by the action of cis-diammine dichloroplatinum (II). Most cross-linked proteins were components of the nuclear matrix, and their heterogeneity reflected the different complexity of liver and erythrocytes matrices, respectively. Some basic proteins, including histones, were also cross-linked, particularly in erythrocyte nuclei. South-Western blotting revealed that a variety of proteins isolated from the cross-linked liver nuclei recognized DNA specifically. In this group of proteins two relatively abundant, acidic, species of 38 and 66 kDa, respectively, might represent novel DNA-binding proteins from the nuclear matrix. In the case of erythrocytes, only the basic proteins showed a DNA-recognition capacity, and among them there were some unidentified species, absent from liver. Lamin B2 was cross-linked but was unable to recognize DNA, and the same was true for other abundant, cross-linked proteins from both types of nuclei. This led to the hypothesis that for some DNA-nuclear matrix interactions the aggregation typical of matrix proteins is essential for the specificity of DNA recognition. Hybridization analysis of the DNA isolated from the cross-linked complexes showed that SARs (scaffold attachment regions) and telomeric sequences were well represented in the cross-linked fragments, that the cross-linked DNA of liver was partially different from that of erythrocytes and that two defined SAR sequences were found to be present only in the cross-linked DNA. These results are in agreement with the present views on DNA-nuclear matrix interactions, which are usually studied on isolated nuclear matrices or purified proteins. Instead, our results provide experimental evidence obtained directly from intact nuclei. © 1996 Wiley-Liss, Inc. 相似文献
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MFP1, a novel plant filament-like protein with affinity for matrix attachment region DNA. 总被引:9,自引:1,他引:8
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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 regulation of gene expression. Here, we report a novel nuclear matrix-localized MAR DNA binding protein, designated MAR binding filament-like protein 1 (MFP1), from tomato. In contrast to the few animal MAR DNA binding proteins thus far identified, MFP1 contains a predicted N-terminal transmembrane domain and a long filament-like alpha-helical domain that is similar to diverse nuclear and cytoplasmic filament proteins from animals and yeast. DNA binding assays established that MFP1 can discriminate between animal and plant MAR DNAs and non-MAR DNA fragments of similar size and AT content. Deletion mutants of MFP1 revealed a novel, discrete DNA binding domain near the C terminus of the protein. MFP1 is an in vitro substrate for casein kinase II, a nuclear matrix-associated protein kinase. Its structure, MAR DNA binding activity, and nuclear matrix localization suggest that MFP1 is likely to participate in nuclear architecture by connecting chromatin with the nuclear matrix and potentially with the nuclear envelope. 相似文献