Purification and properties of theDrosophila zen protein

Summary The zen protein is encoded by the zerknullt gene required for normal early development inDrosophila. Like many regulatory proteins of this type, zen contains a 60 amino acid homeobox sequence. We have purified the zen protein and studied its solution behavior and its interaction with DNA. The zen protein exists as a monomer in solution with a molecular weight of about 40000. It binds specifically to a site about 900 bases upstream from thezen gene. Within this binding site DNase protection experiments indicate that binding is confined to two regions approximately 11 and 14 bases in length that are separated by about 30 base pairs. The protein concentration dependence of the binding curve suggests that protein binding is non cooperative.     

HMG protein binding to an A/T-rich positive regulatory region of the pea plastocyanin gene promoter

Gel retardation assays using pea nuclear extracts have detected specific binding to regions of the promoter of the pea plastocyanin gene (petE). Several complexes which differ in sensitivity to competition with unlabelled promoter fragments and various DNA alternating copolymers, to heat treatment and to digestion with proteinase K have been detected. A protein factor, PCF1, forming one of these complexes was heat-stable and most sensitive to competition with poly(dAdT).poly(dAdT) compared to other alternating copolymers. DNase I footprinting assays showed that tracts of A/T-rich sequence within the -444 to -177 positive regulatory region of the petE promoter were protected in the presence of the pea nuclear extract. The factor PCF1 copurified with a high-mobility-group (HMG) protein preparation from pea chromatin. DNase I footprinting with the HMG protein preparation demonstrated that similar tracts of A/T-rich sequences within the promoter were protected. Southwestern-blot analysis of pea HMG proteins purified by gel filtration through Superose 12 detected a single DNA-binding species of 21 kDa. The properties of the factor PCF1 suggest that it is likely to be an HMG I protein.     

Detection of two tissue-specific DNA-binding proteins with affinity for sites in the mouse beta-globin intervening sequence 2.

To identify proteins from uninduced murine erythroleukemia nuclear extracts which specifically bind to sequences from the DNase I-hypersensitive region within the mouse beta-globin intervening sequence 2 (IVS2), a gel electrophoretic mobility shift assay was used. Two distinct sequence-specific binding proteins were detected. The specific binding sites for these factors were delineated by both DNase I protection footprinting and methylation interference. Factor B1 bound specifically to two homologous sites, B1-A and B1-B, approximately 100 base pairs apart within the IVS2 and on opposite strands. These two regions could interact with factor B1 independently. Factor B1 was limited to cells of hematopoietic lineages. Factor B2 bound to a site approximately 5 base pairs away from the B1-A site and was limited to cells of the erythroid lineage. The limited tissue distribution of these factors and the locations of their binding sites suggest that one or both of these factors may be involved in the formation of the tissue-specific DNase I-hypersensitive site in the IVS2 of the mouse beta-globin gene.     

The interplay of ubiquitous DNA-binding factors, availability of binding sites in the chromatin, and DNA methylation in the differential regulation of phosphoenolpyruvate carboxykinase gene expression.

We have identified DNA elements in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter which are bound 'in vivo' by proteins under conditions of basal level gene expression and have evaluated several hypothesis to account for the tissue specific expression of the gene. In vitro DNase I footprinting demonstrated that factors which bind to basal expression elements of the PEPCK promoter, the BSE/CRE and NFI/CCAAT sites, are also present in HTC and XC cells which do not express the PEPCK gene. 'In vivo' DNase I footprinting demonstrated that the BSE/CRE, NFI/CCAAT, and three additional sites are bound by protein in H4IIE cells which express the PEPCK gene but not in the HTC or XC cells. No evidence for a repressor protein or for phased nucleosome binding to the PEPCK promoter in HTC or XC cells could be detected. Genomic sequencing was used to determine if differential methylation of the PEPCK promoter could account for the lack of factor binding in HTC and XC nuclei. None of the 14 cytosine residues in CpG dinucleotides was methylated in H4IIE or rat liver DNA, all were methylated in rat sperm DNA, and 6 were methylated in HTC DNA; including the cytosine at position--90 within the BSE/CRE. Only one cytosine residue, at position--90, was methylated in XC DNA. Treatment of XC cells with 5-azacytidine resulted in loss of methylation at the--90 position yet this was insufficient to allow synthesis of a detectable amount of PEPCK mRNA.     

Binding of ArsR, the repressor of the Staphylococcus xylosus (pSX267) arsenic resistance operon to a sequence with dyad symmetry within the ars promoter

arsR, the first gene of the Staphylococcus xylosus (pSX267) arsenic/antimonite resistance (rs) operon encodes a negative regulatory protein, ArsR, which mediates inducibility of the resistances by arsenic and antimony compounds. ArsR, which has no obvious DNA-binding motif in its primary structure, was purified from an ArsR-overproducing Escherichia coli strain and identified as a DNA-binding protein by its behaviour in gel mobility shift assays. ArsR had a specific affinity for a 312 by DNA restriction fragment carrying the ars promoter; the minimum sequence complexed by ArsR was a 75 by polymerase chain reaction (PCR) fragment, which mainly comprised the –35 and –10 regions of the promoter. The effect of inducers on the DNA-binding activity of ArsR was examined by in vitro induction assays; only arsenite inhibited DNA-binding of the repressor. DNase I footprinting revealed two protected regions within the promoter region, spanning 23 and 9 nucleotides, respectively. Furthermore, a new cleavage site for DNase I between the protected regions was made accessible by binding of the repressor. The footprints cover a region of three inverted repeats located between the –35 and –10 motifs of the ars promoter. By high resolution footprinting with the hydroxy radical, five sites of close contact between the protein and DNA were identified.     

Binding sites for maize nuclear proteins in the terminal inverted repeats of the Mul transposable element

Summary Nuclear protein extracts from Mu-active, Mu-inactive and non-Mutator lines of maize were used to identify the binding sites for maize nuclear proteins in the terminal inverted repeats (TIR) of the Mul transposable element. We found binding activities of nuclear proteins that specifically interact with both TIRs of the Mu1 element. DNase I footprinting was performed to localize the binding sites. We found that the nuclear proteins from Mu-active lines and non-Mu lines bound to the Mu1 TIR at two different sites, i.e. a 13 by sequence (CGGGAACGGTAAA, designated as site I) and another 8 by sequence (CGGCGTCT, designated as site II). However, the nuclear proteins from Mu-inactive lines bound only one of these sites, i.e. site I. Mobility shift assays with synthetic oligonucleotides containing site I and 11 respectively confirmed the specificities of these binding activities. Site I was shown to be an imperfect direct repeat of a hexamer binding site (CGGGAA CGGTAA). Oligonucleotides containing either of the hexamers showed specific binding activity to nuclear protein from both Mu-active and Mu-inactive lines. The possible role of these proteins in Mu transposition is discussed.     

Determination of equilibrium binding affinity of distamycin and netropsin to the synthetic deoxyoligonucleotide sequence d(GGTATACC)2 by quantitative DNase I footprinting

A new method for determining the equilibrium binding constant of antitumor drugs to specific DNA sequences by quantitative DNase I footprinting is presented. The use of a short synthetic DNA oligomer to define a homogeneous population of DNA binding sites enables the calculation of the free drug concentration and the fraction of DNA sites complexed with drug in solution and is described for the first time. Since a 1:1 stoichiometry is observed for each drug-oligomer DNA complex, it becomes possible to calculate equilibrium binding constants in solution. By use of this technique, the binding affinities of the nonintercalating drugs netropsin and distamycin to the synthetic oligonucleotide d(GGTATACC)2 are determined to be Ka (25 degrees C) = 1.0 X 10(5) and 2.0 X 10(5) M-1, respectively. Quantitation of the temperature dependence associated with complex formation results in a determination of standard enthalpies of -3.75 and -8.48 kcal mol-1 for the binding of netropsin and distamycin, respectively. Calculation of other thermodynamic parameters are found to be in agreement with previous studies and indicate that the DNA binding process for these compounds is predominantly enthalpy driven. This method of quantitative DNase I footprinting is demonstrated to be a useful technique for the measurement of drug affinities to specific binding sites on DNA oligomers which are designed and synthesized expressly for this purpose. Applications of the technique to the determination of drug binding affinities at specific sites within native DNA sequences are discussed.     

Multiple interactions between nuclear proteins of Zea mays and the promoter of the Shrunken gene

Summary Nuclear proteins were extracted from isolated nuclei of immature maize kernels. The promoter region (1.5 kb) of the Shrunken gene, which is highly transcribed in the developing endosperm of the kernel, was scanned for protein-DNA interactions. Several promoter fragments showed protein-DNA complex formation in gel retardation experiments. Two different nucleo-protein complexes (MNP1 and MNP2) have been distinguished in competition and DNase I footprinting experiments. Both nuclear DNA-binding activities are able to recognize multiple sites distributed over a 1.5 kb upstream region of the Shrunken gene. Some of the binding sites established in the in vitro reconstitution experiments are located near to DNase I hypersensitive sites found in the promoter of the Shrunken gene (Frommer and Starlinger 1988).     

An upstream regulatory element of the NCAM promoter contains a binding site for homeodomains.

In the present study, we have analyzed an upstream regulatory element of the neural cell adhesion molecule (NCAM) promoter which is required for full promoter activity. It contains an ATTATTA motif that resembles the core recognition sequence of homeodomain (HD) proteins of the Antennapedia (Antp) and related types. Electrophoretic mobility shift (EMSA) and DNase I footprinting analyses revealed that the Drosophila HDs coded by the Antp and the zerknüllt (zen) genes bind this site in vitro. In contrast, the engrailed (en) protein did not produce a detectable footprint. The functional relevance of the ATTATTA motif was demonstrated by showing that a two-nucleotide exchange curtailed stimulation of an heterologous promoter. An oligonucleotide known to be recognized with high affinity by Antp-like HDs efficiently competed for endogenous factor binding. These results suggest that the NCAM gene may be a target for HD proteins.     

Protein-binding sites within the 5'' DNase I-hypersensitive region of the chicken alpha D-globin gene.

We mapped at high resolution and as a function of development the hypersensitive domain in the 5'-flanking region of the chicken alpha D-globin gene and determined the specific protein-binding sites within the domain. The domain extends from -130 to +80 nucleotides (nt) relative to the cap site. DNase I footprinting within intact embryonic erythrocyte nuclei revealed a strongly protected area from -71 to -52 nt. The same area was weakly protected in adult nuclei. A factor was present in extracts of erythrocyte nuclei from both embryos and adults that protected the sequence AAGATAAGG (-63 to -55 nt) in DNase I footprinting experiments; at higher concentrations of extract, sequences immediately adjacent (-73 to -64 and -53 to -38) were also protected. The same pattern of binding was revealed by gel mobility shift assays. The identical AAGATAAGG sequence is found in the 5'-flanking region of the beta rho gene; it competed for binding of the alpha D-specific factor, suggesting that regulatory elements are shared.     

Interaction of the lymphocyte-derived Epstein-Barr virus nuclear antigen EBNA-1 with its DNA-binding sites.

The Epstein-Barr virus (EBV) nuclear antigen EBNA-1 plays an integral role in the maintenance of latency in EBV-infected B lymphocytes. EBNA-1 binds to sequences within the plasmid origin of replication (oriP). It is essential for the replication of the latent episomal form of EBV DNA and may also regulate the expression of the EBNA group of latency gene products. We have used sequence-specific DNA-binding assays to purify EBNA-1 away from nonspecific DNA-binding proteins in a B-lymphocyte cell extract. The availability of this eucaryotic protein has allowed an examination of the interaction of EBNA-1 with its specific DNA-binding sites and an evaluation of possible roles for the different binding loci within the EBV genome. DNA filter binding assays and DNase I footprinting experiments showed that the intact Raji EBNA-1 protein recognized the two binding site loci in oriP and the BamHI-Q locus and no other sites in the EBV genome. Competition filter binding experiments with monomer and multimer region I consensus binding sites indicated that cooperative interactions between binding sites have relatively little impact on EBNA-1 binding to region I. An analysis of the binding parameters of the Raji EBNA-1 to the three naturally occurring binding loci revealed that the affinity of EBNA-1 for the three loci differed. The affinity for the sites in region I of oriP was greater than the affinity for the dyad symmetry sites (region II) of oriP, while the physically distant region III locus showed the lowest affinity. This arrangement may provide a mechanism whereby EBNA-1 can lowest affinity. This arrangement may provide a mechanism whereby EBNA-1 can mediate differing regulatory functions through differential binding to its recognition sequence.     

Mode of DNA binding by SopA protein of Escherichia coli F plasmid

The binding of SopA to the promoter region of its own gene, in which four copies of SopA's recognition sequence, 5'-CTTTGC-3', are arrayed asymmetrically, was examined in vitro. Titration using electrophoretic mobility shift assay showed that the stoichiometry of SopA protomers to the promoter-region DNA is 4 and that the binding is highly co-operative. The co-operativity was corroborated by EMSA and DNase I footprinting for a number of mutant DNA fragments in which 5'-CTTTGC-3' was changed to 5'-CTTACG-3'. EMSA in the style of circular permutation showed that SopA bends DNA. Mutation at either outermost binding site had a different effect on DNA bending by SopA, reflecting the asymmetry in the arrangement of the binding sites, for which the results of DNase I footprinting were in agreement. Gel filtration chromatography and analytical ultracentrifugation of free SopA showed that the protein can exist as a monomer and oligomers in the absence of ATP. Hence, the results indicate that the co-operativity in SopA's DNA binding is based on its intrinsic protein-protein interaction modified by DNA interaction.