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.     

Nuclear matrix proteins bind very tightly to specific regions of the chicken histone H5 gene.

The nuclear matrix is operationally defined as the structure remaining after nuclease-digested nuclei are extracted with high concentrations of salt. The nuclear matrix is thought to have a role in organizing higher order chromatin into loop domains. We determined whether specific regions of the histone H5 gene were very tightly bound to protein of erythrocyte and liver nuclear matrices in vitro. We demonstrate that DNA fragments spanning sequences 5' to the promoter and the 3' enhancer region of the histone H5 gene, but not DNA fragments spanning the promoter, were very tightly bound to protein of nuclear matrices of erythrocytes and liver. The nuclear matrix consists of internal nuclear matrix and nuclear pore-lamina complex. Recently, we demonstrated that histone deacetylase could be used as a marker enzyme of the internal nuclear matrix. We demonstrate that nuclear pore-lamina complex preparations that were depleted of histone deacetylase activity, and thus of internal nuclear matrix, retained the protein that bound very tightly to the beta-globin and histone H5 enhancers. These results provide evidence that specific regions of the histone H5 gene are very tightly bound to nuclear pore-lamina complex protein.     

The same nuclear proteins bind the proximal CACCC box of the human beta-globin promoter and a similar sequence in the enhancer

Using in vitro assays, we show that nuclear proteins related to the Sp1 and GT-1 factors bind to a CACCC box sequence in the human beta-globin enhancer, adjacent to binding sites for the erythroid-specific factor NFE1 and the ubiquitous factor CP1. The same proteins are known to bind to the proximal, but not to the distal, CACCC, box in the human beta-globin promoter. A C G mutation in the promoter CACCC box, known to cause beta-thalassemia, greatly decreases protein binding to the CACCC box; the same effect is obtained when this mutation is introduced into the enhancer CACCC box.     

Complexes of nuclear matrix DNA with proteins tightly bound to DNA contain a specific small-size RNA of a novel type

Analysis of DNA-protein structures composed of nuclear matrix attached DNA and the most tightly bound proteins was performed. Although the previously described non-histone proteins (1) were present the buoyant density of the complex was the same as that of pure DNA. RNA inaccessible to RNase in 0.4 M NaCl but digestible in low ionic strength buffer was detected. This RNA is not a nascent one. It turned out to be homogeneous and represent a novel type of small nuclear RNA. Partial sequence of this RNA is presented.     

In vitro characterization of tissue-specific nuclear proteins preferentially bound to the mouse beta-globin gene during MEL cell terminal differentiation

Using DNA restriction fragments of the mouse beta-globin gene and other promoter-containing DNA fragments (LTR-MMTV and pBR322) as controls, we have characterized by protein blotting, in extracts of mouse erythroleukemia (MEL) cells, specific nuclear DNA binding proteins with a preferential affinity for the beta-globin DNA. Some proteins (110 and 75 kDa) appear in differentiated MEL cells while others (100, 95, and 35 kDa) are present in immature MEL and normal erythroblast cells and bind selectively to the far-upstream region of the gene. These proteins could modulate either positively or negatively the expression of the beta-globin gene and maybe, of other genes, during the terminal differentiation of erythroid cells.     

Proteins tightly bound to HeLa cell DNA at nuclear matrix attachment sites.

DNA-protein complexes have been isolated from HeLa cell nuclei and nuclear matrix preparations. Two proteins, 55 and 66 kilodaltons in size, remain bound to HeLa DNA after treatment at 80 degrees C in 2% sodium dodecyl sulfate and purification by exclusion chromatography on Sepharose 2B-CL in the presence of 0.3% sodium dodecyl sulfate. These proteins appear to be tightly bound but not covalently linked to the DNA, and they are distributed over the DNA with an average spacing of 40 kilobase pairs. This spacing distribution remains essentially constant throughout the cell cycle. The proteins are bound to the residual 2% of HeLa cell DNA which remains attached to the nuclear matrix after extensive nuclease digestion, a condition which reduces the average size of the DNA to approximately 150 base pairs. Our results suggest that these tightly bound proteins are involved in anchoring cellular DNA to the nuclear matrix. These tightly bound proteins are identical by partial peptide mapping to proteins found tightly bound to the DNA of mammalian, plant, and bacterial cells (D. Werner and C. Petzelt, J. Mol. Biol. 150:297-302, 1981), implying that these proteins are involved in the organization of chromosomal domains and are highly conserved in both procaryotic and eucaryotic cells.     

Binding in vitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA.

Seven protein-binding sites on the immunoglobulin heavy-chain (IgH) enhancer element have been identified by exonuclease III protection and gel retardation assays. It appears that the seven sites bind a minimum of four separate proteins. Three of these proteins also bind to other enhancers or promoters, but one protein seems to recognize exclusively IgH enhancer sequences. A complex of four binding sites, recognized by different proteins, is located within one 80-base-pair region of IgH enhancer DNA. Close juxtaposition of enhancer proteins may allow protein-protein interactions or be part of a mechanism for modulating enhancer protein activity. All IgH enhancer-binding proteins identified in this study were found in extracts from nonlymphoid as well as lymphoid cells. These data provide the first direct evidence that multiple proteins bind to enhancer elements and that while some of these proteins recognize common elements of many enhancers, others have more limited specificities.     

At least two nuclear proteins bind specifically to the Rous sarcoma virus long terminal repeat enhancer.

We used the sensitive gel electrophoresis DNA-binding assay and DNase I footprinting to detect at least two protein factors (EFI and EFII) that bound specifically to the Rous sarcoma virus (RSV) enhancer in vitro. These factors were differentially extracted from quail cell nuclei, recognized different nucleotide sequences in the U3 region of the RSV long terminal repeat, and appeared to bind preferentially to opposite DNA strands as monitored by the DNase I protection assay. The EFI- and EFII-protected regions within U3 corresponded closely to sequences previously demonstrated by deletion mutagenesis to be required for enhancer activity, strongly suggesting a functional significance for these proteins. Only weak homologies between other enhancer consensus sequence motifs and the EFI and EFII recognition sites were observed, and other viral enhancers from simian virus 40 and Moloney murine sarcoma virus did not compete effectively with the RSV enhancer for binding either factor.     

Identification of cellular differentiation-dependent nuclear factors that bind to a human gene for thymidylate synthase.

Three nuclear factors were identified that interact with sequences in the 5'-upstream region of the human thymidylate synthase gene. Two of these factors interact with a sequence around the initiation codon of the thymidylate synthase gene. The amounts of these two factors changed dramatically as human promyelocytic leukemia HL-60 cells differentiated into macrophage-like cells by the treatment with 1,25-dihydroxyvitamin D3. The change was closely correlated with the decrease in the amount of thymidylate synthase mRNA during the differentiation. These findings suggest that the specific nuclear factors are involved in the regulation of the expression of human thymidylate synthase gene during the differentiation of HL-60 cells.     

DNA fragments which specifically bind to isolated nuclear matrix in vitro interact with matrix-associated DNA topoisomerase II

A matrix-associated region (MAR)-containing fragment has been selected from the library of cloned chicken nuclear matrix-associated DNA fragments. Factors, which determine the specific binding of DNA fragments have been studied. Using topoisomerase II-specific inhibitor VM 26 we established that nuclear matrix-associated topoisomerase II interacted with the MAR-containing DNA fragment producing specific cleavage sites on DNA of the fragment.     

Antibodies to the most tightly bound proteins in eukaryotic DNA

DNA released from eukaryotic cells by proteases/SDS or by alkali/SDS still contains distinct proteins which are not removed by these cell lysis procedures nor by subsequent phenol treatment. The proteins most tightly bound to DNA can only be isolated by degradation of DNA. In contrast to the protein-DNA complexes, the protein material isolated after degradation of DNA is sensitive to protease treatment. Moreover, the isolated protein material tends to form aggregates which are insoluble in buffers not containing detergents. They are only poorly soluble in buffers containing SDS. The partially solubilized material can be separated by SDS-polyacrylamide gel electrophoresis into two main bands. Antibodies were raised in rabbits against the polypeptides contained in these main bands. Immunofluorescence micrographs are presented of cells treated with the antibodies. The results indicate that the proteins characterized by their involvement in extremely stable protein-DNA complexes also occur independently of DNA in eukaryotic cells.     

Interaction of various nuclear matrix proteins with DNA

An attempt has been made to localize the previously detected strong and weak bonds of nuclear matrix proteins with DNA in some groups of proteins, using fractionation of the matrix into lamina and intranuclear fibrils, isolation of the "elementary globules", fractionation of matrix nucleoproteins on hydroxyapatite. It was shown that both weak and strong bonds are localized on the nuclear lamina and in the intranuclear fibrils. The single-stranded DNA enriched fraction of the matrix nucleoproteins contained mostly strong bonds. The strong bond is less resistant to pronase treatment. A method for isolating nuclear matrix nucleoprotein fractions carrying only strong or only weak bonds is proposed.