Stimulates DNA Binding Research Articles

The pentapeptide motif of Hox proteins is required for cooperative DNA binding with Pbx1, physically contacts Pbx1, and enhances DNA binding by Pbx1.

The vertebrate Hox genes, which represent a subset of all homeobox genes, encode proteins that regulate anterior-posterior positional identity during embryogenesis and are cognates of the Drosophila homeodomain proteins encoded by genes composing the homeotic complex (HOM-C). Recently, we demonstrated that multiple Hox proteins bind DNA cooperatively with both Pbx1 and its oncogenic derivative, E2A-Pbx1. Here, we show that the highly conserved pentapeptide motif F/Y-P-W-M-R/K, which occurs in numerous Hox proteins and is positioned 8 to 50 amino acids N terminal to the homeodomain, is essential for cooperative DNA binding with Pbx1 and E2A-Pbx1. Point mutational analysis demonstrated that the tryptophan and methionine residues within the core of this motif were critical for cooperative DNA binding. A peptide containing the wild-type pentapeptide sequence, but not one in which phenylalanine was substituted for tryptophan, blocked the ability of Hox proteins to bind cooperatively with Pbx1 or E2A-Pbx1, suggesting that the pentapeptide itself provides at least one surface through which Hox proteins bind Pbx1. Furthermore, the same peptide, but not the mutant peptide, stimulated DNA binding by Pbx1, suggesting that interaction of Hox proteins with Pbx1 through the pentapeptide motif raises the DNA-binding ability of Pbx1.

McrB: a prokaryotic protein specifically recognizing DNA containing modified cytosine residues.

Restriction of DNA by the Escherichia coli K-12 McrBC restriction endonuclease, which consists of the two subunits McrB and McrC, depends on the presence of modified cytosine residues in a special constellation. From previous work by others it was known that restriction of 5-methylcytosine-containing DNA requires two methylated 5'-PuC sites separated by approximately 40-80 non-defined base pairs. Here we show that binding of the McrBC nuclease is mediated exclusively by the McrB subunit. McrB has a low affinity for non-methylated DNA, with which it forms low molecular weight complexes. The affinity for DNA is significantly increased, with variations depending on the sequence context, by hemi- or fully methylated 5'-PuC sites. Binding to such substrates yields high molecular weight complexes, presumably involving several McrB molecules. Methylation at unique 5'-PuC sites can be sufficient to stimulate DNA binding by McrB. As such substrates are not cleaved by the nuclease, restriction apparently requires the coordinated interaction of molecules bound to neighbouring 5'-PumC sites. The binding properties of McrB exhibit some similarities to recently identified eukaryotic proteins interacting in a non-sequence-specific manner with DNA containing methylated 5'-CpG sequences and might point to a common molecular origin of these proteins. In addition to DNA, McrB also binds GTP, an essential cofactor in DNA restriction by McrBC. McrC neither binds to DNA nor modulates the DNA binding potential of McrB. As McrC is essential for restriction it appears to predominantly function in catalysis.

Sodium Salicylate and Yeast Heat Shock Gene Transcription

The anti-inflammatory drug sodium salicylate modulates the activity of specific transcription factors in humans. Here we show that salicylate and sorbate, another organic acid, stimulate DNA binding by yeast heat shock transcription factor (HSF) in vivo. Surprisingly, salicylate inhibits heat shock gene transcription even in cells induced by a prior heat shock. This inhibition of transcription occurs at a step after HSF and transcription factor IID binding but before promoter melting by RNA polymerase. Salicylate appears to generate a tight binding but activation-impotent HSF by cytoplasmic acidification, since inhibiting proton efflux from cells triggers this same DNA binding and inhibition of heat shock gene expression.

Developmental expression of APEX nuclease, a multifunctional DNA repair enzyme, in mouse brains

Expression of the mammalian major apurinic/apyrimidinic (AP) endonuclease (designated as APEX nuclease, or HAPI, APE or Ref-1 gene product) during mouse brain development was investigated by in situ and northern blot hybridizations. The enzyme is known to be a redox factor (Ref-1) stimulating DNA binding activity of AP-1 binding proteins such as Fos and Jun as well as a multifunctional DNA repair enzyme having 5′ AP endonuclease, DNA 3′ repair diesterase, 3′–5′ exonuclease and DNA 3′-phosphatase activities. In the embryonic and postnatal development, APEX mRNA was expressed at high levels in the proliferative zone of various brain regions, with showing temporal and spatial changes. Its expression decreased in association with brain development to the basal expression level which was observed even in adulthood, with the exception of its expression in the hippocampal formation. The growth dependent expression of APEX gene suggests that it has some roles on cell proliferation and/or differentiation in developmental brain. Its expression on the hippocampal formation became significant from postnatal day 7 and then increased. The pyramidal and granule cell layers expressed it at a higher level than most other brain regions at postnatal day 21. The developmental change of APEX gene expression was not necessarily associated with the changes of expression of c- fos and c- jun genes measured by northern blot hybridization. However, the present results suggested that APEX/Ref-1 gene product can interact with AP-1 binding proteins in brain, especially in the hippocampal formation, to regulate some brain functions by redox-activation.

The protein kinase C activator, phorbol ester, cooperates with the wild-type p53 species of Ras-transformed embryo fibroblasts growth arrest.

Clone 112 cells, a rat embryo fibroblast cell line cotransfected by an activated ras gene and a temperature-sensitive mutant p53 gene (p53val135) grow well at 37 degrees C but cease DNA synthesis and cell division when shifted to 32 degrees C (Michalovitz, D., Halevy, O., and Oren, M. (1990) Cell 62, 671-680). Characterization of the p53 protein in exponentially growing clone 112 cells at 37 degrees C revealed that both wild-type (reactive with the monoclonal antibody PAb 246) and mutant (reactive with PAb 240) p53 conformational forms are co-expressed. These results indicate that in clone 112 cells the growth suppressor activity of the wild-type p53 species is inactivated at 37 degrees C. We show that clone 112 cells grown at 37 degrees C elicits specific growth inhibition response to stimulation by the tumor promoter phorbol ester, phorbol 12-myristate 13-acetate (PMA). At 37 degrees C, PMA induced nuclear accumulation of the p53 protein, a behavior that is also observed in growth-arrested cells at 32 degrees C. Furthermore, when cells are growth arrested at 32 degrees C, PMA prevented the cells from re-entering the cell cycle when they are shifted back to 37 degrees C. All these observations suggest that PMA can cooperate with the wild-type p53 in cell growth arrest. At 37 and 32 degrees C, PMA stimulation of clone 112 cells resulted in specific enhancement of phosphorylation of the wild-type p53 species but not of the mutant form. We also demonstrate that the growth arrest of clone 112 cells at 37 degrees C is correlated with stimulation of the nuclear wild-type p53-DNA binding activities. The PMA-mediated increase in p53 DNA binding activity coincides with the loss of the PAb 421 epitope on the p53.DNA complex. PAb 421 non-reactivity with p53 has been shown by others to occur in growth-arrested cells and upon phosphorylation of p53 by protein kinase C. We also provide evidence that, in vitro, the protein kinase C mode of phosphorylation stimulates DNA binding activities of purified recombinant wild-type p53 and that in a mutant conformation p53 is not a substrate for protein kinase C. We propose that wild-type p53 and protein kinase C, the cellular receptor of phorbol ester, could participate in the negative feedback controls associated with the phosphoinositide-derived signals common to a number of mitogenic stimulations.

Sphingosine 1-phosphate, a novel signaling molecule, stimulates DNA binding activity of AP-1 in quiescent Swiss 3T3 fibroblasts.

Sphingosine and sphingosine 1-phosphate, metabolites of sphingolipids, stimulate cell proliferation in quiescent Swiss 3T3 fibroblasts and induce transient increases in intracellular free calcium (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell Biol. 114, 155-167). However, little is yet known of the nuclear events that follow the early responses induced by sphingolipid metabolites. Using a gel retardation assay, we found that specific DNA binding activity of activator protein-1 (AP-1) was markedly increased after treatment of quiescent Swiss 3T3 fibroblasts with sphingosine 1-phosphate and sphingosine. The DNA binding specificity of AP-1 was confirmed with competing probes containing consensus sequences of AP-1, AP-2, AP-3, SP-1, and NF1/CTF. The c-fos gene product was detected in the AP-1 complex using anti-c-Fos antibody. The dose response for stimulation of DNA binding activity of AP-1 by sphingosine 1-phosphate correlated closely with its effect on DNA synthesis. Furthermore, an inhibitor of sphingosine kinase, DL-threo-dihydrosphingosine, which inhibits sphingosine-induced DNA synthesis and the formation of sphingosine 1-phosphate, also inhibited sphingosine-stimulated AP-1 DNA binding activity. This result further supports our proposal that sphingosine 1-phosphate mediates the mitogenic effect of sphingosine. Our results indicate that sphingosine 1-phosphate-induced DNA synthesis and cell division may result from activation of AP-1 protein, linking signal transduction by sphingolipid metabolites to gene expression.

HTLV-I Tax protein stimulation of DNA binding of bZIP proteins by enhancing dimerization.

The Tax protein of human T cell leukemia virus type-1 (HTLV-I) transcriptionally activates the HTLV-I promoter. This activation requires binding sites for activating transcription factor (ATF) proteins, a family of cellular proteins that contain basic region-leucine zipper (bZIP) DNA binding domains. Data are presented showing that Tax increases the in vitro DNA binding activity of multiple ATF proteins. Tax also stimulated DNA binding by other bZIP proteins, but did not affect DNA binding proteins that lack a bZIP domain. The increase in DNA binding occurred because Tax promotes dimerization of the bZIP domain in the absence of DNA, and the elevated concentration of the bZIP homodimer then facilitates the DNA binding reaction. These results help explain how Tax activates viral transcription and transforms cells.

A cellular factor stimulates the DNA-binding activity of MyoD and E47.

We show that mixing purified MyoD and E47 proteins results in heterodimers that fail to bind DNA, even though MyoD and E47 homodimers can bind DNA efficiently. Addition of cell extracts or a specific fraction from a cell extract enables the heterodimer to bind DNA, but components in this fraction fail to enter the DNA complex. The activity is sensitive to heat and protease and is not ATP-dependent. The activity functions on E47 and MyoD homodimers and can stimulate DNA binding of the basic-helix-loop-helix region of MyoD. The effectiveness of the activity, for MyoD homodimers, depends on the exact DNA sequence of the binding site. Our results suggest that specific factors in the cell might control the DNA-binding properties of helix-loop-helix proteins.

Thioredoxin regulates the DNA binding activity of NF-kappa B by reduction of a disulphide bond involving cysteine 62.

A role for redox regulation in activation of the NF-kappa B transcription factor was suggested by the observation that DNA binding activity of free protein, but not preformed DNA-protein complex, is inhibited by -SH modifying agents but enhanced by reducing agents. Mutagenesis of conserved cysteine residues in the p50 subunit identified amino acid 62 as being important for DNA binding, as a serine substitution at this position reduces DNA binding affinity, but renders the protein insensitive to -SH modifying agents. DNA binding activity of the wild type protein but not the amino acid 62 mutant was also stimulated by thioredoxin while detection of disulphide cross linked dimers in p50 but not the amino acid 62 mutant suggests that thioredoxin stimulates DNA binding by reduction of a disulphide bond involving cysteine 62. The physiological relevance of these findings was supported by the observation that cotransfection of a plasmid expressing human thioredoxin and an HIV LTR driven reporter construct resulted in an NF-kappa B dependent increase in expression of the reporter gene. Thus modification of p50 by thioredoxin, a gene induced by stimulation of T-lymphocytes in parallel with NF-kappa B translocation, is a likely step in the cascade of events leading to full NF-kappa B activation.

Ecdysterone Receptor Is a Sequence-Specific Transcription Factor Involved in the Developmental Regulation of Heat Shock Genes

Purification of ecdysterone receptor from Drosophila melanogaster to apparent homogeneity is reported. Purified receptor binds specifically to several sequences in the promoters of the developmentally active hsp27 and hsp23 heat shock genes that were previously implied in ecdysterone regulation of the genes and that share limited homology among themselves and with mammalian steroid receptor binding sites. Some of these elements confer ecdysterone regulation on a basal promoter in transfected cells, acting in a synergistic fashion. Transcription in vitro of promoters containing such elements is stimulated up to 100-fold by added purified ecdysterone receptor, depending on receptor dosage and the number of elements present. Transcriptional enhancement requires sequence-specific binding of receptor to template promoters which facilitates the formation of a preinitiation complex. Ecdysterone stimulates DNA binding of the receptor in vitro.

Ecdysterone receptor is a sequence-specific transcription factor involved in the developmental regulation of heat shock genes.

Purification of ecdysterone receptor from Drosophila melanogaster to apparent homogeneity is reported. Purified receptor binds specifically to several sequences in the promoters of the developmentally active hsp27 and hsp23 heat shock genes that were previously implied in ecdysterone regulation of the genes and that share limited homology among themselves and with mammalian steroid receptor binding sites. Some of these elements confer ecdysterone regulation on a basal promoter in transfected cells, acting in a synergistic fashion. Transcription in vitro of promoters containing such elements is stimulated up to 100-fold by added purified ecdysterone receptor, depending on receptor dosage and the number of elements present. Transcriptional enhancement requires sequence-specific binding of receptor to template promoters which facilitates the formation of a preinitiation complex. Ecdysterone stimulates DNA binding of the receptor in vitro.

Activation of the heat shock transcription factor by hypoxia in mammalian cells.

Members of the stress protein family such as HSP70 are induced in ischemic tissues and may contribute to the ability of cells to survive episodes of transient circulatory insufficiency. However, the biochemical events that lead to this induction, and their degree of similarity with pathways triggered by heat stress, have not been defined. In this study, we demonstrate that transient exposure of cultured C2C12 mouse myogenic cells to a hypoxic atmosphere stimulates DNA binding activity of the heat shock transcription factor through mechanisms that are independent of new protein synthesis. Activation of heat shock transcription factor in hypoxic cells is temporally associated with induction of endogenous HSP70 gene transcription and with induction of a heterologous reporter gene controlled by the human HSP70 promoter. Furthermore, induction of the human HSP70 promoter by hypoxia requires an intact heat shock element, indicating that other cis-acting transcriptional control elements contained within this complex promoter are not sufficient to transduce signals generated within hypoxic cells. These findings provide strong evidence that hypoxia and heat shock induce expression of the HSP70 gene by similar, if not identical, mechanisms.

The thyroid hormone receptor binds to multiple domains of the rat growth hormone 5′-flanking sequence.

Gel shift assays have been employed to examine the association of the thyroid hormone receptor with specific DNA sequences in the 5'-flanking DNA of the rat growth hormone (rGH) gene. This DNA is known to have structure(s) that mediate thyroid hormone effects on the rGH promoter. The receptors used were obtained from preparations purified 300-500-fold from rat liver nuclear extracts and contained about 1% pure receptors. Thyroid hormone receptor binding to DNA was assessed by monitoring protein-bound 32P-labeled restriction endonuclease fragments in parallel with L-tri[125I]iodothyronine-labeled protein-DNA complexes. The receptors were found to bind specifically to four different regions of the rGH 5'-flanking DNA (nucleotides -1730 to -1230, -530 to -230, -181 to -149, and -149 to +12) numbered with respect to the transcriptional start site. The specificity of the binding was documented by the finding that the receptor did not bind to other rGH 5'-flanking DNA sequences or to several other DNAs and by the fact that only the DNAs exhibiting specific binding could block the binding of radiolabeled DNA. The binding was also detected in NaCl concentrations up to 140 mM, reduced by Mg2+ concentrations up to 5 mM, and inhibited by 1 mM zinc. The DNA sequence-specific binding of the receptor was found to require occupancy of the receptor by the hormone (L-triiodothyronine) and could also be observed when the receptor was occupied by the thyroid hormone antagonist amiodarone. These results indicate that thyroid hormone receptors interact specifically with several sites on the 5'-flanking DNA of the rGH gene and that hormone occupancy is not required for the binding. Thus, thyroid hormone may act by stimulating a transcriptional activation function of the receptor rather than by stimulating DNA binding per se.

The heat-stable cytosolic factor that promotes glucocorticoid receptor binding to DNA is neither thioredoxin nor ribonuclease.

Treatment of rat liver cytosol containing temperature-transformed [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). However, transformed receptors that are treated with MMTS and then separated from low Mr components of cytosol by passage through a column of Sephadex G-50 have very little DNA-binding activity when DTT is added to regenerate sulfhydryl moities. The receptors will bind to DNA if whole liver cytosol or boiled liver cytosol is added in addition to DTT. The effect of boiled cytosol is mimicked by purified rat thioredoxin or bovine RNase A in a manner that does not reflect the reducing activity of the former or the catalytic activity of the latter. This suggests that the reported ability of each of these heat-stable peptides to stimulate DNA binding by glucocorticoid receptors is not a biologically relevant action. We suggest that stimulation of DNA binding of partially purified receptors by boiled cytosol does not constitute a reconstitution of a complete cytosolic system in which the dissociated receptor must associate with a specific heat-stable accessory protein required for DNA binding, as has been suggested in the "two-step" model of receptor transformation recently proposed by Schmidt et al. (Schmidt T.J., Miller-Diener, A., Webb M.L. and Litwack G. (1985) J. biol. Chem. 260, 16255-16262).

DNA Binding and Uptake by Nuclei Isolated from Plant Protoplasts

Binding and uptake of exogenous DNA by nuclei isolated from Glycine max L. Merr were studied using (3)H-labeled single-stranded DNA of bacteriophage fd. A comparison of single-stranded with double-stranded DNA for binding and uptake by nuclei was also made.Isolated nuclei were incubated with (3)H-labeled single-stranded bacteriophage fd DNA. Poly-l-lysine or DEAE-dextran at 0.1 and 1 mug/ml stimulated DNA binding. On the other hand, poly-l-lysine at 1 and 5 mug/ml increased DNA uptake but DEAE-dextran did not. Ten to 20 mm Ca and Mg ions were required for DNA uptake. EDTA (1-20 mm) did not differ from the control or the low levels of Ca or Mg ions. These observations on single-stranded fd DNA differed from those obtained with double-stranded DNA of Salmonella typhimurium. Kinetics of single-stranded DNA binding and uptake also deviated from that of double-stranded DNA.Analyses of nuclei-bound DNA by sucrose density gradient and CsCI density gradient centrifugation revealed extensive DNA degradation during a 45-minute incubation period. Poly-l-lysine protected against rapid degradation of bound DNA. DEAE-dextran enhanced DNA binding, but bound DNA was cleaved into much smaller polymers than those detected in control experiments or in the presence of poly-l-lysine. Sucrose density gradient and CsCI density gradient centrifugation analyses on DNA taken up by nuclei also showed extensive DNA degradation. Poly-l-lysine slightly inhibited DNA degradation, but DEAE-dextran appeared to enhance degradation of incorporated DNA. Moreover, nonbound DNA in the incubation medium was completely degraded within a 20-minute incubation period.

DNA Binding and Uptake by Nuclei Isolated from Plant Protoplasts

DNA binding and uptake by nuclei isolated from soybean (Glycine max L. Merr.) protoplasts were investigated using radioactive homogeneous DNA prepared from soybean cells. DNA binding to nuclei was found to decrease drastically with increased incubation time. Total uptake and acid-precipitable uptake reached a maximum after 20 minutes of incubation. Optimum DNA binding and uptake occurred at pH 6 and the process was enhanced by increasing the incubation temperature to 40 C. Salmonella typhimurium DNA and poly ([dA-dT]-[dA-dT]) competitively inhibited DNA binding whereas calf thymus DNA was less competitive; however, Micrococcus lysodeikticus DNA stimulated DNA binding and tobacco mosaic virus RNA had no effect. DNA binding and uptake was enhanced by addition of Mg ions, Ca ions, poly-l-lysine, and ATP. Increasing amounts of EDTA appeared to decrease DNA binding. Pronase strongly inhibited DNA binding and uptake.